Pirin polypeptide and immune modulation

ABSTRACT

The present invention relates to polypeptide HP, or a polynucleotide sequence encoding polypeptide HP, or a host cell comprising said polynucleotide sequence, or a host cell comprising an expression vector comprising said polynucleotide sequence, for use in the treatment and/or prevention of a disorder in a subject; wherein said disorder is an inflammatory disorder and/or an autoimmune disorder; wherein said polypeptide has at least 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 or variants, homologues, fragments or derivatives thereof; and wherein said polynucleotide sequence encodes a polypeptide which has at least 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 or variants, homologues, fragments or derivatives thereof and/or wherein said polynucleotide sequence has at least 75% identity to SEQ ID NO 1, SEQ ID NO 3 or SEQ ID NO 5 or variants, homologues, fragments or derivatives thereof.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 7, 2016, isnamed p067638WO_sequence_listing.txt and is 14,000 bytes in size.

CROSS-REFERENCE

This application is a continuation of International Application No.PCT/GB2015/054113, filed Dec. 22, 2015, which claims the benefit ofGreat Britain Application No. 1423083.3, filed Dec. 23, 2014, which arehereby incorporated by reference in their entirety.

FIELD OF INVENTION

The present invention relates to the polypeptide HP or a polynucleotidesequence encoding polypeptide HP or a host cell comprising saidpolynucleotide sequence or a host cell comprising an expression vectorcomprising said polynucleotide sequence for various therapeutic andnutritional uses.

BACKGROUND

Bacteroides thetaiotaomicron has potent anti-inflammatory effects invitro and in vivo (Kelly et al. Commensal anaerobic gut bacteriaattenuate inflammation by regulating nuclear-cytoplasmic shuttling ofPPAR-gamma and RelA. Nat Immunol. 2004 January; 5(1):104-12). Itmodulates molecular signalling pathways of NF-κB (Kelly et al, Commensalanaerobic gut bacteria attenuate inflammation by regulatingnuclear-cytoplasmic shuttling of PPAR-gamma and RelA. Nat Immunol. 2004January; 5(1):104-12). In particular, it stops binding of the activecomponent (RelA) of NF-κB to key genes in the nucleus, therebypreventing the activation of pro-inflammatory pathways (Kelly et al,Commensal anaerobic gut bacteria attenuate inflammation by regulatingnuclear-cytoplasmic shuttling of PPAR-gamma and RelA. Nat Immunol. 2004January; 5(1):104-12).

The full genome of B. thetaiotaomicron was sequenced and annotated bythe Gordon Group (Washington University School of Medicine, USA) in 2003[Xu et al, A genomic view of the human-Bacteroides thetaiotaomicronsymbiosis. Science. 2003 Mar. 28; 299(5615):2074-6].

STATEMENTS OF INVENTION

Surprisingly, the present inventors found that HP (a hypotheticalprotein; gene ID 1075517; gene symbol BT_0187; accession numberAA075294) identified from the genome of Bacteroides thetaiotaomicron(VPI5482), a pirin-related protein; deposited as AAO75294.1, whichreduces inflammation in cells.

The present invention provides polypeptide HP, or a polynucleotidesequence encoding polypeptide HP, or a host cell comprising saidpolynucleotide sequence, or a host cell comprising an expression vectorcomprising said polynucleotide sequence, for use in the treatment and/orprevention of a disorder in a subject; wherein said disorder is aninflammatory disorder and/or an autoimmune disorder; wherein saidpolypeptide has at least 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQID NO 6 or variants, homologues, fragments or derivatives thereof; andwherein said polynucleotide sequence encodes a polypeptide which has atleast 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 orvariants, homologues, fragments or derivatives thereof and/or whereinsaid polynucleotide sequence has at least 75% identity to SEQ ID NO 1,SEQ ID NO 3 or SEQ ID NO 5 or variants, homologues, fragments orderivatives thereof.

In another aspect, the present invention provides polypeptide HP, or apolynucleotide sequence encoding polypeptide HP, or a host cellcomprising said polynucleotide sequence, or a host cell comprising anexpression vector comprising said polynucleotide sequence, for use inmodulating the inflammation of a cell, a tissue or an organ in asubject; wherein said polypeptide has at least 75% identity to SEQ ID NO2, SEQ ID NO 4 or SEQ ID NO 6 or variants, homologues, fragments orderivatives thereof; and wherein said polynucleotide sequence encodes apolypeptide which has at least 75% identity to SEQ ID NO 2, SEQ ID NO 4or SEQ ID NO 6 or variants, homologues, fragments or derivatives thereofand/or wherein said polynucleotide sequence has at least 75% identity toSEQ ID NO 1, SEQ ID NO 3 or SEQ ID NO 5 or variants, homologues,fragments or derivatives thereof.

In a further aspect, the present invention provides polypeptide HP, or apolynucleotide sequence encoding polypeptide HP, or a host cellcomprising said polynucleotide sequence, or a host cell comprising anexpression vector comprising said polynucleotide sequence, for use inimproving intestine barrier integrity in a subject; wherein saidpolypeptide has at least 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQID NO 6 or variants, homologues, fragments or derivatives thereof; andwherein said polynucleotide sequence encodes a polypeptide which has atleast 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 orvariants, homologues, fragments or derivatives thereof and/or whereinsaid polynucleotide sequence has at least 75% identity to SEQ ID NO 1,SEQ ID NO 3 or SEQ ID NO 5 or variants, homologues, fragments orderivatives thereof.

The present invention provides, in another aspect, polypeptide HP, or apolynucleotide sequence encoding polypeptide HP, or a host cellcomprising said polynucleotide sequence, or a host cell comprising anexpression vector comprising said polynucleotide sequence, for use inmodifying the bacterial composition in a tissue or organ to provide amore beneficial microbiota. For example, the invention may be of use inreducing the level of one or more types of lactose fermenting bacteria(such as E. coli) in a tissue or an organ in a subject and/or reducingthe level of one or more types of non-lactose fermenting bacteria in atissue or an organ in a subject; wherein said polypeptide has at least75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 or variants,homologues, fragments or derivatives thereof; and wherein saidpolynucleotide sequence encodes a polypeptide which has at least 75%identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 or variants,homologues, fragments or derivatives thereof and/or wherein saidpolynucleotide sequence has at least 75% identity to SEQ ID NO 1, SEQ IDNO 3 or SEQ ID NO 5 or variants, homologues, fragments or derivativesthereof.

The present invention provides, in a further aspect, polypeptide HP, ora polynucleotide sequence encoding polypeptide HP, or a host cellcomprising said polynucleotide sequence, or a host cell comprising anexpression vector comprising said polynucleotide sequence, for use inmaintaining the length of the large intestine and/or small intestine ofa subject, (e.g. said subject has IBD); wherein said polypeptide has atleast 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 orvariants, homologues, fragments or derivatives thereof; and wherein saidpolynucleotide sequence encodes a polypeptide which has at least 75%identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 or variants,homologues, fragments or derivatives thereof and/or wherein saidpolynucleotide sequence has at least 75% identity to SEQ ID NO 1, SEQ IDNO 3 or SEQ ID NO 5 or variants, homologues, fragments or derivativesthereof.

In a further aspect, the present invention provides polypeptide HP, or apolynucleotide sequence encoding polypeptide HP, or a host cellcomprising said polynucleotide sequence, or a host cell comprising anexpression vector comprising said polynucleotide sequence, for use inreducing disruption to the intestine (such as the large intestine) of asubject, (e.g. said subject has IBD); wherein said polypeptide has atleast 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 orvariants, homologues, fragments or derivatives thereof; and wherein saidpolynucleotide sequence encodes a polypeptide which has at least 75%identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 or variants,homologues, fragments or derivatives thereof and/or wherein saidpolynucleotide sequence has at least 75% identity to SEQ ID NO 1, SEQ IDNO 3 or SEQ ID NO 5 or variants, homologues, fragments or derivativesthereof.

In another aspect, the present invention provides polypeptide HP, or apolynucleotide sequence encoding polypeptide HP, or a host cellcomprising said polynucleotide sequence, or a host cell comprising anexpression vector comprising said polynucleotide sequence, for use inregulating the expression of one or more pro-inflammatory genes and/orone or more barrier integrity genes in a cell or cells of a subject;wherein said polypeptide has at least 75% identity to SEQ ID NO 2, SEQID NO 4 or SEQ ID NO 6 or variants, homologues, fragments or derivativesthereof; and wherein said polynucleotide sequence encodes a polypeptidewhich has at least 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO6 or variants, homologues, fragments or derivatives thereof and/orwherein said polynucleotide sequence has at least 75% identity to SEQ IDNO 1, SEQ ID NO 3 or SEQ ID NO 5 or variants, homologues, fragments orderivatives thereof.

The present invention provides, in another aspect, polypeptide HP, or apolynucleotide sequence encoding polypeptide HP, or a host cellcomprising said polynucleotide sequence, or a host cell comprising anexpression vector comprising said polynucleotide sequence, for use inregulating the expression in a cell or cells of a subject of one or moregenes selected from the group consisting of regenerating islet-derived 3beta gene (Reg3b), resistin-like gamma resistin like beta gene(Retnlg|Retnlb), sucrase-isomaltase (alpha-glucosidase) gene (Si),defensin alpha 24 gene (Defa24), hydroxysteroid 11-beta dehydrogenase 2gene (Hsd11b2), hydroxysteroid (17-beta) dehydrogenase 2 gene (Hsd17b2),resistin-Like Molecule-beta (RELMb), and nuclear receptor 1D1 thyroidhormone receptor alpha gene (Nr1d1|Thra); (e.g. said subject has IBD);wherein said polypeptide has at least 75% identity to SEQ ID NO 2, SEQID NO 4 or SEQ ID NO 6 or variants, homologues, fragments or derivativesthereof; and wherein said polynucleotide sequence encodes a polypeptidewhich has at least 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO6 or variants, homologues, fragments or derivatives thereof and/orwherein said polynucleotide sequence has at least 75% identity to SEQ IDNO 1, SEQ ID NO 3 or SEQ ID NO 5 or variants, homologues, fragments orderivatives thereof.

In a further aspect, the present invention provides polypeptide HP or apolynucleotide sequence encoding polypeptide HP, or a host cellcomprising said polynucleotide sequence, or a host cell comprising anexpression vector comprising said polynucleotide sequence, for use inreducing the activation of pro-inflammatory pathways in a cell or cellsof a subject; wherein said polypeptide has at least 75% identity to SEQID NO 2, SEQ ID NO 4 or SEQ ID NO 6 or variants, homologues, fragmentsor derivatives thereof; and wherein said polynucleotide sequence encodesa polypeptide which has at least 75% identity to SEQ ID NO 2, SEQ ID NO4 or SEQ ID NO 6 or variants, homologues, fragments or derivativesthereof and/or wherein said polynucleotide sequence has at least 75%identity to SEQ ID NO 1, SEQ ID NO 3 or SEQ ID NO 5 or variants,homologues, fragments or derivatives thereof.

The present invention provides, in a further aspect, polypeptide HP, ora polynucleotide sequence encoding polypeptide HP, or a host cellcomprising said polynucleotide sequence, or a host cell comprising anexpression vector comprising said polynucleotide sequence, for use inreducing the activity and/or expression of NF-κβ in a cell or cells(such as epithelial cells, epidermal cells, neuronal cells, and/orpancreatic cells) of a subject; wherein said polypeptide has at least75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 or variants,homologues, fragments or derivatives thereof; and wherein saidpolynucleotide sequence encodes a polypeptide which has at least 75%identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 or variants,homologues, fragments or derivatives thereof and/or wherein saidpolynucleotide sequence has at least 75% identity to SEQ ID NO 1, SEQ IDNO 3 or SEQ ID NO 5 or variants, homologues, fragments or derivativesthereof.

The present invention provides, in another aspect, polypeptide HP, or apolynucleotide sequence encoding polypeptide HP, or a host cellcomprising said polynucleotide sequence, or a host cell comprising anexpression vector comprising said polynucleotide sequence, for use inimproving alimentary canal health in a subject; wherein said polypeptidehas at least 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 orvariants, homologues, fragments or derivatives thereof; and wherein saidpolynucleotide sequence encodes a polypeptide which has at least 75%identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 or variants,homologues, fragments or derivatives thereof and/or wherein saidpolynucleotide sequence has at least 75% identity to SEQ ID NO 1, SEQ IDNO 3 or SEQ ID NO 5 or variants, homologues, fragments or derivativesthereof.

The present invention provides, in a further aspect, a pharmaceuticalcomposition comprising polypeptide HP, or a polynucleotide sequenceencoding polypeptide HP, or a host cell comprising said polynucleotidesequence, or a host cell comprising an expression vector comprising saidpolynucleotide sequence, and a pharmaceutically acceptable excipient,carrier or diluent; wherein said polypeptide has at least 75% identityto SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 or variants, homologues,fragments or derivatives thereof; and wherein said polynucleotidesequence encodes a polypeptide which has at least 75% identity to SEQ IDNO 2, SEQ ID NO 4 or SEQ ID NO 6 or variants, homologues, fragments orderivatives thereof and/or wherein said polynucleotide sequence has atleast 75% identity to SEQ ID NO 1, SEQ ID NO 3 or SEQ ID NO 5 orvariants, homologues, fragments or derivatives thereof.

In a further aspect, the present invention provides a nutritionalsupplement comprising polypeptide HP, or a polynucleotide sequenceencoding polypeptide HP, or a host cell comprising said polynucleotidesequence, or a host cell comprising an expression vector comprising saidpolynucleotide sequence, and a nutritional acceptable excipient, carrieror diluent; wherein said polypeptide has at least 75% identity to SEQ IDNO 2, SEQ ID NO 4 or SEQ ID NO 6 or variants, homologues, fragments orderivatives thereof; and wherein said polynucleotide sequence encodes apolypeptide which has at least 75% identity to SEQ ID NO 2, SEQ ID NO 4or SEQ ID NO 6 or variants, homologues, fragments or derivatives thereofand/or wherein said polynucleotide sequence has at least 75% identity toSEQ ID NO 1, SEQ ID NO 3 or SEQ ID NO 5 or variants, homologues,fragments or derivatives thereof.

In another aspect, the present invention provides a feedstuff, foodproduct, dietary supplement, or food additive comprising polypeptide HP,or a polynucleotide sequence encoding polypeptide HP, or a host cellcomprising said polynucleotide sequence, or a host cell comprising anexpression vector comprising said polynucleotide sequence; wherein saidpolypeptide has at least 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQID NO 6 or variants, homologues, fragments or derivatives thereof; andwherein said polynucleotide sequence encodes a polypeptide which has atleast 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 orvariants, homologues, fragments or derivatives thereof and/or whereinsaid polynucleotide sequence has at least 75% identity to SEQ ID NO 1,SEQ ID NO 3 or SEQ ID NO 5 or variants, homologues, fragments orderivatives thereof.

The present invention provides in a further aspect a process forproducing a pharmaceutical composition according to the presentinvention, said process comprising admixing polypeptide HP, or apolynucleotide sequence encoding polypeptide HP, or a host cellcomprising said polynucleotide sequence, or a host cell comprising anexpression vector comprising said polynucleotide sequence with apharmaceutically acceptable excipient, carrier or diluent; optionallysaid polypeptide or polynucleotide sequence or host cell is encapsulatedin said process; wherein said polypeptide has at least 75% identity toSEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 or variants, homologues,fragments or derivatives thereof; and wherein said polynucleotidesequence encodes a polypeptide which has at least 75% identity to SEQ IDNO 2, SEQ ID NO 4 or SEQ ID NO 6 or variants, homologues, fragments orderivatives thereof and/or wherein said polynucleotide sequence has atleast 75% identity to SEQ ID NO 1, SEQ ID NO 3 or SEQ ID NO 5 orvariants, homologues, fragments or derivatives thereof.

In a further aspect, the present invention provides a process forproducing a nutritional supplement according to the present invention,said process comprising admixing polypeptide HP, or a polynucleotidesequence encoding polypeptide HP, or a host cell comprising saidpolynucleotide sequence, or a host cell comprising an expression vectorcomprising said polynucleotide sequence with a nutritionally acceptableexcipient, carrier or diluent; optionally said polypeptide orpolynucleotide is encapsulated in said process; wherein said polypeptidehas at least 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 orvariants, homologues, fragments or derivatives thereof; and wherein saidpolynucleotide sequence encodes a polypeptide which has at least 75%identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 or variants,homologues, fragments or derivatives thereof and/or wherein saidpolynucleotide sequence has at least 75% identity to SEQ ID NO 1, SEQ IDNO 3 or SEQ ID NO 5 or variants, homologues, fragments or derivativesthereof.

In another aspect, the present invention provides a process forproducing a feedstuff, food product, dietary supplement, or foodadditive according to the present invention, said process comprisingadmixing polypeptide HP, or a polynucleotide sequence encodingpolypeptide HP, or a host cell comprising said polynucleotide sequence,or a host cell comprising an expression vector comprising saidpolynucleotide sequence with a feedstuff, food product, dietarysupplement, food additive or ingredient thereof; optionally saidpolypeptide or polynucleotide is encapsulated in said process; whereinsaid polypeptide has at least 75% identity to SEQ ID NO 2, SEQ ID NO 4or SEQ ID NO 6 or variants, homologues, fragments or derivativesthereof; and wherein said polynucleotide sequence encodes a polypeptidewhich has at least 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO6 or variants, homologues, fragments or derivatives thereof and/orwherein said polynucleotide sequence has at least 75% identity to SEQ IDNO 1, SEQ ID NO 3 or SEQ ID NO 5 or variants, homologues, fragments orderivatives thereof.

The present invention provides, in another aspect, a method for treatingand/or preventing a disorder in a subject, wherein said method comprisesadministering to the subject polypeptide HP, or a polynucleotidesequence encoding polypeptide HP, or a host cell comprising saidpolynucleotide sequence, or a host cell comprising an expression vectorcomprising said polynucleotide sequence; wherein said polypeptide orpolynucleotide sequence or host cell treats and/or prevents a disorderin the subject, wherein said disorder is an inflammatory disorder and/oran autoimmune disorder; wherein said polypeptide has at least 75%identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 or variants,homologues, fragments or derivatives thereof; and wherein saidpolynucleotide sequence encodes a polypeptide which has at least 75%identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 or variants,homologues, fragments or derivatives thereof and/or wherein saidpolynucleotide sequence has at least 75% identity to SEQ ID NO 1, SEQ IDNO 3 or SEQ ID NO 5 or variants, homologues, fragments or derivativesthereof.

The present invention provides, in a further aspect, a method formodulating the inflammation of a tissue or an organ in a subject whereinsaid method comprises administering to the subject polypeptide HP, or apolynucleotide sequence encoding polypeptide HP, or a host cellcomprising said polynucleotide sequence, or a host cell comprising anexpression vector comprising said polynucleotide sequence; wherein saidpolypeptide, polynucleotide sequence or host cell modulates theinflammation of a tissue or an organ in the subject; wherein saidpolypeptide has at least 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQID NO 6 or variants, homologues, fragments or derivatives thereof; andwherein said polynucleotide sequence encodes a polypeptide which has atleast 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 orvariants, homologues, fragments or derivatives thereof and/or whereinsaid polynucleotide sequence has at least 75% identity to SEQ ID NO 1,SEQ ID NO 3 or SEQ ID NO 5 or variants, homologues, fragments orderivatives thereof.

In another aspect, the present invention provides a method for improvingintestine barrier integrity in a subject wherein said method comprisesadministering to the subject polypeptide HP, or a polynucleotidesequence encoding polypeptide HP, or a host cell comprising saidpolynucleotide sequence, or a host cell comprising an expression vectorcomprising said polynucleotide sequence; wherein said polypeptide orpolynucleotide sequence or host cell improves intestine barrierintegrity in the subject; wherein said polypeptide has at least 75%identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 or variants,homologues, fragments or derivatives thereof; and wherein saidpolynucleotide sequence encodes a polypeptide which has at least 75%identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 or variants,homologues, fragments or derivatives thereof and/or wherein saidpolynucleotide sequence has at least 75% identity to SEQ ID NO 1, SEQ IDNO 3 or SEQ ID NO 5 or variants, homologues, fragments or derivativesthereof.

The present invention provides, in another aspect, a method for reducingthe level of one or more types of lactose fermenting bacteria (such asE. coli) in a tissue or an organ in a subject and/or reducing the levelof one or more types of non-lactose fermenting bacteria in a tissue oran organ in a subject, wherein said method comprises administering tothe subject polypeptide HP, or a polynucleotide sequence encodingpolypeptide HP, or a host cell comprising said polynucleotide sequence,or a host cell comprising an expression vector comprising saidpolynucleotide sequence; wherein said polypeptide, polynucleotidesequence or host cell reduces the level of one or more types of lactosefermenting bacteria (such as E. coli) in a tissue or an organ in thesubject and/or reduces the level of one or more types of non-lactosefermenting bacteria in a tissue or an organ in the subject; wherein saidpolypeptide has at least 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQID NO 6 or variants, homologues, fragments or derivatives thereof; andwherein said polynucleotide sequence encodes a polypeptide which has atleast 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 orvariants, homologues, fragments or derivatives thereof and/or whereinsaid polynucleotide sequence has at least 75% identity to SEQ ID NO 1,SEQ ID NO 3 or SEQ ID NO 5 or variants, homologues, fragments orderivatives thereof.

The present invention provides, in a further aspect, a method formaintaining the length of the large intestine and/or small intestine ofa subject wherein said method comprises administering to the subjectpolypeptide HP, or a polynucleotide sequence encoding polypeptide HP, ora host cell comprising said polynucleotide sequence, or a host cellcomprising an expression vector comprising said polynucleotide sequence;(e.g. said subject has IBD); wherein said polypeptide or polynucleotidesequence or host cell maintains the length of the large intestine and/orsmall intestine of the subject; wherein said polypeptide has at least75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 or variants,homologues, fragments or derivatives thereof; and wherein saidpolynucleotide sequence encodes a polypeptide which has at least 75%identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 or variants,homologues, fragments or derivatives thereof and/or wherein saidpolynucleotide sequence has at least 75% identity to SEQ ID NO 1, SEQ IDNO 3 or SEQ ID NO 5 or variants, homologues, fragments or derivativesthereof.

In a further aspect, the present invention provides a method forreducing disruption to the intestine (e.g. the large intestine) of asubject wherein said method comprises administering to the subjectpolypeptide HP, or a polynucleotide sequence encoding polypeptide HP, ora host cell comprising said polynucleotide sequence, or a host cellcomprising an expression vector comprising said polynucleotide sequence;(e.g. said subject has IBD); wherein said polypeptide or polynucleotidesequence or host cell reduces disruption to the intestine (e.g. largeintestine) of the subject; wherein said polypeptide has at least 75%identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 or variants,homologues, fragments or derivatives thereof; and wherein saidpolynucleotide sequence encodes a polypeptide which has at least 75%identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 or variants,homologues, fragments or derivatives thereof and/or wherein saidpolynucleotide sequence has at least 75% identity to SEQ ID NO 1, SEQ IDNO 3 or SEQ ID NO 5 or variants, homologues, fragments or derivativesthereof.

The present invention provides, in another aspect, a method forregulating the expression of one or more pro-inflammatory oranti-inflammatory genes in a cell or cells of a subject wherein saidmethod comprises administering to the subject polypeptide HP, or apolynucleotide sequence encoding polypeptide HP, or a host cellcomprising said polynucleotide sequence, or a host cell comprising anexpression vector comprising said polynucleotide sequence; wherein saidpolypeptide or polynucleotide sequence or host cell regulates theexpression of one or more pro-inflammatory genes and/oranti-inflammatory genes in a cell or cells of the subject; wherein saidpolypeptide has at least 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQID NO 6 or variants, homologues, fragments or derivatives thereof; andwherein said polynucleotide sequence encodes a polypeptide which has atleast 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 orvariants, homologues, fragments or derivatives thereof and/or whereinsaid polynucleotide sequence has at least 75% identity to SEQ ID NO 1,SEQ ID NO 3 or SEQ ID NO 5 or variants, homologues, fragments orderivatives thereof.

In another aspect, the present invention provides a method forregulating the expression of one or more genes in a cell or cells of asubject wherein said method comprises administering to the subjectpolypeptide HP, or a polynucleotide sequence encoding polypeptide HP, ora host cell comprising said polynucleotide sequence, or a host cellcomprising an expression vector comprising said polynucleotide sequence,to said subject; wherein said polypeptide or polynucleotide sequence orhost cell regulates the expression of one or more genes in a cell orcells of the subject; wherein the one or more genes are selected fromthe group consisting of regenerating islet-derived 3 beta gene (Reg3b),resistin-like gamma resistin like beta gene (Retnlg|Retnlb),sucrase-isomaltase (alpha-glucosidase) gene (Si), defensin alpha 24 gene(Defa24), hydroxysteroid 11-beta dehydrogenase 2 gene (Hsd11b2),hydroxysteroid (17-beta) dehydrogenase 2 gene (Hsd17b2), resistin-LikeMolecule-beta (RELMb), and nuclear receptor 1D1 thyroid hormone receptoralpha gene (Nr1d1|Thra); (e.g. said subject has IBD); wherein saidpolypeptide has at least 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQID NO 6 or variants, homologues, fragments or derivatives thereof; andwherein said polynucleotide sequence encodes a polypeptide which has atleast 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 orvariants, homologues, fragments or derivatives thereof and/or whereinsaid polynucleotide sequence has at least 75% identity to SEQ ID NO 1,SEQ ID NO 3 or SEQ ID NO 5 or variants, homologues, fragments orderivatives thereof.

The present invention provides, in a further aspect, a method forreducing the activation of pro-inflammatory pathways in a cell or cellsof a subject wherein said method comprises administering to the subjectpolypeptide HP, or a polynucleotide sequence encoding polypeptide HP, ora host cell comprising said polynucleotide sequence, or a host cellcomprising an expression vector comprising said polynucleotide sequence;wherein said polypeptide or polynucleotide sequence or host cell reducesthe activation of pro-inflammatory pathways in a cell or cells of thesubject; wherein said polypeptide has at least 75% identity to SEQ ID NO2, SEQ ID NO 4 or SEQ ID NO 6 or variants, homologues, fragments orderivatives thereof; and wherein said polynucleotide sequence encodes apolypeptide which has at least 75% identity to SEQ ID NO 2, SEQ ID NO 4or SEQ ID NO 6 or variants, homologues, fragments or derivatives thereofand/or wherein said polynucleotide sequence has at least 75% identity toSEQ ID NO 1, SEQ ID NO 3 or SEQ ID NO 5 or variants, homologues,fragments or derivatives thereof.

In a further aspect, the present invention provides a method forreducing the activity and/or expression of NF-κβ in a cell or cells(such as epithelial cells, epidermal cells, neuronal cells, and/orpancreatic cells) of a subject wherein said method comprisesadministering to the subject polypeptide HP, or a polynucleotidesequence encoding polypeptide HP, or a host cell comprising saidpolynucleotide sequence, or a host cell comprising an expression vectorcomprising said polynucleotide sequence; wherein said polypeptide orpolynucleotide or host cell reduces the activity and/or expression ofNF-κβ in a cell or cells (such as epithelial cells, epidermal cells,neuronal cells, and/or pancreatic cells) of the subject; wherein saidpolypeptide has at least 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQID NO 6 or variants, homologues, fragments or derivatives thereof; andwherein said polynucleotide sequence encodes a polypeptide which has atleast 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 orvariants, homologues, fragments or derivatives thereof and/or whereinsaid polynucleotide sequence has at least 75% identity to SEQ ID NO 1,SEQ ID NO 3 or SEQ ID NO 5 or variants, homologues, fragments orderivatives thereof.

In another aspect, the present invention provides a method for improvingalimentary canal health in a subject wherein said method comprisesadministering to the subject polypeptide HP, or a polynucleotidesequence encoding polypeptide HP, or a host cell comprising saidpolynucleotide sequence, or a host cell comprising an expression vectorcomprising said polynucleotide sequence; wherein said polypeptide orpolynucleotide sequence or host cell improves alimentary canal health inthe subject; wherein said polypeptide has at least 75% identity to SEQID NO 2, SEQ ID NO 4 or SEQ ID NO 6 or variants, homologues, fragmentsor derivatives thereof; and wherein said polynucleotide sequence encodesa polypeptide which has at least 75% identity to SEQ ID NO 2, SEQ ID NO4 or SEQ ID NO 6 or variants, homologues, fragments or derivativesthereof and/or wherein said polynucleotide sequence has at least 75%identity to SEQ ID NO 1, SEQ ID NO 3 or SEQ ID NO 5 or variants,homologues, fragments or derivatives thereof.

The present invention provides, in a further aspect, use of polypeptideHP, or a polynucleotide sequence encoding polypeptide HP, or a host cellcomprising said polynucleotide sequence, or a host cell comprising anexpression vector comprising said polynucleotide sequence, for themanufacture of a medicament for the treatment and/or prevention of adisorder in a subject, wherein said disorder is an inflammatory disorderand/or an autoimmune disorder; wherein said polypeptide has at least 75%identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 or variants,homologues, fragments or derivatives thereof; and wherein saidpolynucleotide sequence encodes a polypeptide which has at least 75%identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 or variants,homologues, fragments or derivatives thereof and/or wherein saidpolynucleotide sequence has at least 75% identity to SEQ ID NO 1, SEQ IDNO 3 or SEQ ID NO 5 or variants, homologues, fragments or derivativesthereof.

The present invention provides, in another aspect, use of polypeptideHP, or a polynucleotide sequence encoding polypeptide HP, or a host cellcomprising said polynucleotide sequence, or a host cell comprising anexpression vector comprising said polynucleotide sequence, for themanufacture of a medicament for modulating the inflammation of a tissueor an organ in a subject; wherein said polypeptide has at least 75%identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 or variants,homologues, fragments or derivatives thereof; and wherein saidpolynucleotide sequence encodes a polypeptide which has at least 75%identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 or variants,homologues, fragments or derivatives thereof and/or wherein saidpolynucleotide sequence has at least 75% identity to SEQ ID NO 1, SEQ IDNO 3 or SEQ ID NO 5 or variants, homologues, fragments or derivativesthereof.

In another aspect, the present invention provides use of polypeptide HPor a polynucleotide sequence encoding polypeptide HP, or a host cellcomprising said polynucleotide sequence, or a host cell comprising anexpression vector comprising said polynucleotide sequence, for themanufacture of a medicament for improving intestine barrier integrity ina subject; wherein said polypeptide has at least 75% identity to SEQ IDNO 2, SEQ ID NO 4 or SEQ ID NO 6 or variants, homologues, fragments orderivatives thereof; and wherein said polynucleotide sequence encodes apolypeptide which has at least 75% identity to SEQ ID NO 2, SEQ ID NO 4or SEQ ID NO 6 or variants, homologues, fragments or derivatives thereofand/or wherein said polynucleotide sequence has at least 75% identity toSEQ ID NO 1, SEQ ID NO 3 or SEQ ID NO 5 or variants, homologues,fragments or derivatives thereof.

The present invention provides, in a further aspect, use of polypeptideHP, or a polynucleotide sequence encoding polypeptide HP, or a host cellcomprising said polynucleotide sequence, or a host cell comprising anexpression vector comprising said polynucleotide sequence, for themanufacture of a medicament for maintaining the length of the largeintestine and/or small intestine of a subject; (e.g. said subject hasIBD); wherein said polypeptide has at least 75% identity to SEQ ID NO 2,SEQ ID NO 4 or SEQ ID NO 6 or variants, homologues, fragments orderivatives thereof; and wherein said polynucleotide sequence encodes apolypeptide which has at least 75% identity to SEQ ID NO 2, SEQ ID NO 4or SEQ ID NO 6 or variants, homologues, fragments or derivatives thereofand/or wherein said polynucleotide sequence has at least 75% identity toSEQ ID NO 1, SEQ ID NO 3 or SEQ ID NO 5 or variants, homologues,fragments or derivatives thereof.

In a further aspect, the present invention provides use of a polypeptideHP, or a polynucleotide sequence encoding polypeptide HP, or a host cellcomprising said polynucleotide sequence, or a host cell comprising anexpression vector comprising said polynucleotide sequence, for themanufacture of a medicament for modifying the bacterial composition in atissue or organ to provide a beneficial microbiota, preferably, for usein reducing the level of one or more types of lactose fermentingbacteria (such as E. coli) in a tissue or an organ in a subject and/orreducing the level of one or more types of non-lactose fermentingbacteria in a tissue or an organ in a subject; wherein said polypeptidehas at least 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 orvariants, homologues, fragments or derivatives thereof; and wherein saidpolynucleotide sequence encodes a polypeptide which has at least 75%identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 or variants,homologues, fragments or derivatives thereof and/or wherein saidpolynucleotide sequence has at least 75% identity to SEQ ID NO 1, SEQ IDNO 3 or SEQ ID NO 5 or variants, homologues, fragments or derivativesthereof.

In another aspect, the present invention provides use of polypeptide HP,or a polynucleotide sequence encoding polypeptide HP, or a host cellcomprising said polynucleotide sequence, or a host cell comprising anexpression vector comprising said polynucleotide sequence, for themanufacture of a medicament for reducing disruption to the intestine(e.g. large intestine) of a subject; (e.g. said subject has IBD);wherein said polypeptide has at least 75% identity to SEQ ID NO 2, SEQID NO 4 or SEQ ID NO 6 or variants, homologues, fragments or derivativesthereof; and wherein said polynucleotide sequence encodes a polypeptidewhich has at least 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO6 or variants, homologues, fragments or derivatives thereof and/orwherein said polynucleotide sequence has at least 75% identity to SEQ IDNO 1, SEQ ID NO 3 or SEQ ID NO 5 or variants, homologues, fragments orderivatives thereof.

The present invention provides, in a further aspect, use of polypeptideHP, or a polynucleotide sequence encoding polypeptide HP, or a host cellcomprising said polynucleotide sequence, or a host cell comprising anexpression vector comprising said polynucleotide sequence, for themanufacture of a medicament for regulating the expression of one or morepro-inflammatory genes in a cell or cells of a subject; wherein saidpolypeptide has at least 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQID NO 6 or variants, homologues, fragments or derivatives thereof; andwherein said polynucleotide sequence encodes a polypeptide which has atleast 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 orvariants, homologues, fragments or derivatives thereof and/or whereinsaid polynucleotide sequence has at least 75% identity to SEQ ID NO 1,SEQ ID NO 3 or SEQ ID NO 5 or variants, homologues, fragments orderivatives thereof.

The present invention provides, in another aspect, use of polypeptideHP, or a polynucleotide sequence encoding polypeptide HP, or a host cellcomprising said polynucleotide sequence, or a host cell comprising anexpression vector comprising said polynucleotide sequence, for themanufacture of a medicament for regulating the expression in a cell orcells of a subject of one or more genes selected from the groupconsisting of regenerating islet-derived 3 beta gene (Reg3b),resistin-like gamma resistin like beta gene (Retnlg|Retnlb),sucrase-isomaltase (alpha-glucosidase) gene (Si), defensin alpha 24 gene(Defa24), hydroxysteroid 11-beta dehydrogenase 2 gene (Hsd11b2),hydroxysteroid (17-beta) dehydrogenase 2 gene (Hsd17b2), resistin-LikeMolecule-beta (RELMb), and nuclear receptor 1D1 thyroid hormone receptoralpha gene (Nr1d1|Thra); (e.g. said subject has IBD); wherein saidpolypeptide has at least 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQID NO 6 or variants, homologues, fragments or derivatives thereof; andwherein said polynucleotide sequence encodes a polypeptide which has atleast 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 orvariants, homologues, fragments or derivatives thereof and/or whereinsaid polynucleotide sequence has at least 75% identity to SEQ ID NO 1,SEQ ID NO 3 or SEQ ID NO 5 or variants, homologues, fragments orderivatives thereof.

In another aspect, the present invention provides use of polypeptide HP,or a polynucleotide sequence encoding polypeptide HP, or a host cellcomprising said polynucleotide sequence, or a host cell comprising anexpression vector comprising said polynucleotide sequence, for themanufacture of a medicament for reducing the activation ofpro-inflammatory pathways in a cell or cells of a subject; wherein saidpolypeptide has at least 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQID NO 6 or variants, homologues, fragments or derivatives thereof; andwherein said polynucleotide sequence encodes a polypeptide which has atleast 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 orvariants, homologues, fragments or derivatives thereof and/or whereinsaid polynucleotide sequence has at least 75% identity to SEQ ID NO 1,SEQ ID NO 3 or SEQ ID NO 5 or variants, homologues, fragments orderivatives thereof.

In a further aspect, the present invention provides use of a polypeptideHP, or a polynucleotide sequence encoding polypeptide HP, or a host cellcomprising said polynucleotide sequence, or a host cell comprising anexpression vector comprising said polynucleotide sequence, for themanufacture of a medicament for reducing the activity and/or expressionof NF-κβ in a cell or cells (such as epithelial cells, epidermal cells,neuronal cells, and/or pancreatic cells) of a subject; wherein saidpolypeptide has at least 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQID NO 6 or variants, homologues, fragments or derivatives thereof; andwherein said polynucleotide sequence encodes a polypeptide which has atleast 75% identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 orvariants, homologues, fragments or derivatives thereof and/or whereinsaid polynucleotide sequence has at least 75% identity to SEQ ID NO 1,SEQ ID NO 3 or SEQ ID NO 5 or variants, homologues, fragments orderivatives thereof.

The present invention provides, in another aspect, use of a polypeptideHP, or a polynucleotide sequence encoding polypeptide HP, or a host cellcomprising said polynucleotide sequence, or a host cell comprising anexpression vector comprising said polynucleotide sequence, for themanufacture of a medicament for improving alimentary canal health in asubject.

FIGURES

The invention is described with reference to the accompanying figures,wherein:

FIG. 1A shows an alignment of the polynucleotide sequences encoding HP(SEQ ID NO 1), E. coli optimised HP (Rec 1 HP—SEQ ID NO 3) and L. lactisoptimised HP (Rec 2 HP—SEQ ID NO 5). HP is deposited with GenBank underaccession number: AAO75294.1 and is described in GenBank as possiblePirin family protein [Bacteroides thetaiotaomicron VPI-5482].

FIG. 1B shows an alignment of the polypeptide sequences HP (SEQ ID NO2), E. coli optimised HP (Rec 1 HP—SEQ ID NO 4) and L. lactis optimisedHP (Rec 2 HP—SEQ ID NO 6). HP is deposited with GenBank under accessionnumber: AAO75294.1 and is described in GenBank as possible Pirin familyprotein [Bacteroides thetaiotaomicron VPI-5482].

FIG. 2 shows the change in weight, water and food intakes by rats givenDextran Sodium Sulphate (DSS) in water with (DSS/HP) or without (DSS)co-treatment with hypothetical protein (HP).

FIG. 3 shows the length of the colon and small intestine in rats givenDextran Sodium Sulphate in water with (DSS/HP) or without (DSS)co-treatment with hypothetical protein (HP).

FIG. 4 shows the numbers of lactose-fermenting (predominantly E. coli)and non-lactose-fermenting bacteria in tissues from rats given DextranSodium Sulphate (DSS) in water with (DSS/HP) or without (DSS)co-treatment with hypothetical protein (HP). [When Log10=1.0, nobacteria were detected].

FIG. 5 shows the morphology of the ascending and descending colon fromrats given Dextran Sodium Sulphate in water with (DSS/HP) or without(DSS) co-treatment with hypothetical protein (HP).

FIG. 6 shows the mean histopathology scores and histopathology scores aspercentage fields of view with pathology of grades 0-3 for the ascendingcolon from rats given Dextran Sodium Sulphate in water with (DSS/HP) orwithout (DSS) co-treatment with hypothetical protein (HP).

FIG. 7 shows the heatmap of 377 differentially expressed genes in tissuefrom rats given Dextran Sodium Sulphate in water with or withoutco-treatment with hypothetical protein (HP).

FIG. 8 shows the expression of inflammation-associated genes (RealtimePCR) in ascending colons from rats given Dextran Sodium Sulphate inwater with (DSS/HP) or without (DSS) co-treatment with hypotheticalprotein (HP).

DETAILED DESCRIPTION

HP

Without wishing to be bound by theory, the polypeptide HP of the presentinvention is a pirin-related protein.

Polypeptide HP has at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or100% identity to the polypeptide sequence shown as SEQ ID NO 2, SEQ IDNO 4 or SEQ ID NO 6 or variants, homologues, fragments or derivativesthereof.

One example of polypeptide HP of the present invention is SEQ ID NO 2deposited with GenBank as AAO75294.1; Bacteroides thetaiotaomicroncomprising SEQ ID NO 1 can be found deposited as DSM2079 [E50(VPI5482),VPI5482] at DSMZ (Deutsche Sammlung von Mikroorganismen and ZellkulturenGmbH). The polypeptide sequence SEQ ID NO 2 has the following sequence:

        10         20         30         40MKKVIDRASS RGYFNHGWLK THHTFSFANY YNPERIHFGA        50         60         70         80LRVLNDDSVD PSMGFDTHPH KNMEVISIPL KGYLRHGDSV        90        100        110        120QNTKTITPGD IQVMSTGSGI YHSEYNDSKE EQLEFLQIWV       130        140        150        160FPRIENTKPE YNNFDIRPLL KPNELSLFIS PNGKTPASIK       170        180        190        200QDAWFSMGDF DTERTIEYCM HQEGNGAYLF VIEGEISVAD       210        220        230 EHLAKRDGIG IWDTKSFSIR ATKGTKLLVM EVPM

AAO75294.1 is described as being a possible Pirin family protein.AAO75294.1 was identified from Bacteroides thetaiotaomicron VPI-5482.

The polypeptides sequences deposited in GenBank as AAO76683.1 andCDE80552.1 are examples of polypeptides having at least 75% identity toSEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6.

The polypeptide sequence of AAO76683.1 is as follows:

        10         20         30         40MKKVIHKADT RGHSQYDWLD SYHTFSFDEY FDSDRINFGA        50         60         70         80LRVLNDDKVA PGEGFQTHPH KNMEIISIPL KGHLQHGDSK        90        100        110        120KNSRIITVGE IQTMSAGTGI FHSEVNASPV EPVEFLQIWI       130        140        150        160MPRERNTHPV YKDFSIKELE RPNELAVIVS PDGSTPASLL       170        180        190        200QDTWFSIGKV EAGKKLGYHL HQSHGGVYIF LIEGEIVVDG       210        220        230 EVLKRRDGMG VYDTKSFELE TLKDSHILLI EVPM

The polypeptide sequence of AAO76683.1 is also referred to as SEQ ID NO7 herein. AAO76683.1 is described in GenBank as being a putative Pirinfamily protein. AAO76683.1 was isolated from Bacteroidesthetaiotaomicron VPI-5482. [E50(VPI5482), VPI5482]. Bacteroidesthetaiotaomicron comprising SEQ ID NO 7 can be found deposited asDSM2079 BT 1576 at DSMZ (Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH).

The polypeptide sequence of CDE80552.1 is as follows:

        10         20         30         40MKKVIHKADT RGHSQYDWLD SYHTFSFDEY FDSDRINFGA        50         60         70         80LRVLNDDKVA PGEGFQTHPH KNMEIISIPL KGHLQHGDSK        90        100        110        120KNSRIITVGE IQTMSAGTGI FHSEVNASPV EPVEFLQIWI        130        140        150        160MPRERNTHPV YKDFSIKELE RPNELAVIVS PDGSTPASLL       170        180        190        200QDTWFSIGKV EAGKKLGYHL HQSHGGVYIF LIEGEIVVDG       210        220        230 EVLKRRDGMG VYDTKSFELE TLKDSHILLI EVPM

The polypeptide sequence of CDE80552.1 is also referred to as SEQ ID NO8 herein. CDE80552.1 is described in GenBank as being a putative Pirinfamily protein. CDE80552.1 was isolated from Bacteroidesthetaiotaomicron CAG:40.

The terms “polypeptide HP”, “HP polypeptide” and “HP” are usedinterchangeably herein.

In one embodiment, the polypeptide HP is the polypeptide shown as SEQ IDNO 2.

In another embodiment, the polypeptide HP is the polypeptide shown asSEQ ID NO 4.

In further embodiment, the polypeptide HP is the polypeptide shown asSEQ ID NO 6.

HP polypeptides can be derived from certain microorganisms. In oneaspect, the HP polypeptide is derived from an anaerobic, gram negativebacterium which can live in the alimentary canal. In a further aspect,the HP polypeptide is derived from a Bacteroides spp such as aBacteroides thetaiotaomicron.

Examples of a polynucleotide sequence encoding polypeptide HP includethe polynucleotide sequences shown as SEQ ID NO 1, SEQ ID NO 3 or SEQ IDNO 5; polynucleotide sequences encoding the polypeptide shown as SEQ IDNO 2, SEQ ID NO 4 or SEQ ID NO 6; polynucleotides sequences having atleast 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO 1,SEQ ID NO 3 or SEQ ID NO 5 or variants, homologues, fragments orderivatives thereof; polynucleotides sequences encoding a polypeptidehaving at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity tothe polypeptide shown as SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6 orvariants, homologues, fragments or derivatives thereof; andpolynucleotide sequences encoding SEQ ID NO 7 or SEQ ID NO 8.

SEQ ID NOs 1, 3 and 5 are shown in FIG. 1A.

SEQ ID NO 1 has the following sequence:

Atgaaaaaagtaatcgacagagcttcatcaagaggctattttaatcatggctggctcaaaacccaccacacattcagttttgctaactattacaatccggaaagaatccatttcggagccttgcgagtgctgaatgatgacagtgtagacccgtcgatgggatttgatactcatccacataaaaatatggaagtaatttccattccgttgaaagggtatctgagacatggcgacagtgtacaaaatacgaaaacgattactcccggtgatatccaagtgatgagtacgggcagtggtatctatcatagtgagtataacgacagcaaggaagaacaattggaattcctgcaaatatgggtattcccccgaatcgagaatacgaaacccgaatataacaatttcgatatacgtccgctgctgaaaccgaacgagttatctctgttcatttcaccgaacggcaagacaccggcctccatcaaacaggatgcctggttctctatgggagacttcgatacggaaagaaccatcgaatattgtatgcatcaggaaggtaacggagcttatctgtttgtgatagaaggagagatcagcgtggccgatgaacatctggccaaacgtgacggcatcggaatatgggataccaaaagcttctctatccgtgctactaaagggaccaaacttctggt aatggaagtacccatgtaa

SEQ ID NO 1 encodes SEQ ID NO 2 which is deposited with GenBank underaccession number AAO75294.1.

The polynucleotide sequence of SEQ ID NO 1 was codon optimised forexpression in E. coli. This codon optimised sequence is shown as SEQ IDNO 3. This sequence may also be referred herein as “Rec 1 HP” or“recombinant 1 HP”.

SEQ ID NO 3 has the following sequence:

ggtaccatgaaaaaagtgattgatcgtgcgagcagccgtggctattttaaccatggctggctgaaaacccatcatacctttagcttcgcgaactattataatccggaacgcattcattttggcgcgctgcgtgtgctgaacgatgatagcgtggatccgagcatgggctttgatacccatccgcacaaaaacatggaagtgattagcattccgctgaaaggctatctgcgtcatggcgatagcgtgcagaacaccaaaaccattaccccgggtgatattcaggtgatgagcaccggcagcggcatttatcatagcgaatacaacgatagcaaagaagaacagctggaatttctgcagatttgggtgtttccgcgtattgaaaacaccaaaccggaatataacaactttgatattcgcccgctgctgaaaccgaacgaactgagcctgtttattagcccgaacggcaaaaccccggcgagcattaaacaggatgcgtggtttagcatgggcgattttgataccgaacgcaccattgaatattgcatgcatcaggaaggcaacggcgcgtacctgtttgtgattgaaggcgaaattagcgtggcggatgaacatctggccaaacgtgatggcattggcatttgggataccaaaagcttcagcattcgtgcgaccaaaggcaccaaactgctggtgatggaag tgccgatgtaataagagctc

The polypeptide sequence encoded by SEQ ID NO 3 is shown as SEQ ID NO 4.

SEQ ID NO 4 has the following sequence:

GTMKKVIDRASSRGYFNHGWLKTHHTFSFANYYNPERIHFGALRVLNDDSVDPSMGFDTHPHKNMEVISIPLKGYLRHGDSVQNTKTITPGDIQVMSTGSGIYHSEYNDSKEEQLEFLQIWVFPRIENTKPEYNNFDIRPLLKPNELSLFISPNGKTPASIKQDAWFSMGDFDTERTIEYCMHQEGNGAYLFVIEGEISVADEHLAKRDGIGIWDTKSFSIRATKGTKLLVMEVPM EL

The polynucleotide sequence of SEQ ID NO 1 was codon optimised forexpression in Lactococcus lactis. This codon optimised sequence is shownas SEQ ID NO 5. This sequence may also be referred herein as “Rec 2 HP”or “recombinant 2 HP”.

SEQ ID NO 5 has the following sequence:

ggtaccatgaaaaaagttattgatcgtgcttcatcacgtggatattttaatcatggatggcttaaaactcatcatacatttagttttgccaattattataatccagaacgtattcattttggtgctcttcgtgttcttaatgatgattcagttgatccatcaatgggatttgatacacatccacataaaaatatggaagttatttcaattccacttaaaggatatcttcgtcatggtgattcagttcaaaatacaaaaacaattacacctggagatattcaagttatgtctacaggatcaggaatttatcattcagaatataatgattcaaaagaagaacaacttgaatttcttcaaatttgggtctttccacgtattgaaaatacaaaaccagaatataataatttcgacattcgtccacttcttaaaccaaatgaactttcactttttatctcaccaaatggaaaaacaccagcttcaattaaacaagatgcttggttttcaatgggagattttgatacagaacgtacaattgaatattgtatgcatcaagaaggtaacggcgcttatctttttgttattgaaggtgaaatttcagttgctgatgaacatcttgctaaacgtgatggaattggaatttgggatacaaaatcattttcaattcgtgctacaaaaggtacaaaacttcttgttatggaag ttccaatgtaataagagctc

The polypeptide sequence encoded by SEQ ID NO 5 is shown as SEQ ID NO 6.

SEQ ID NO 6 has the following sequence:

GTMKKVIDRASSRGYFNHGWLKTHHTFSFANYYNPERIHFGALRVLNDDSVDPSMGFDTHPHKNMEVISIPLKGYLRHGDSVQNTKTITPGDIQVMSTGSGIYHSEYNDSKEEQLEFLQIWVFPRIENTKPEYNNFDIRPLLKPNELSLFISPNGKTPASIKQDAWFSMGDFDTERTIEYCMHQEGNGAYLFVIEGEISVADEHLAKRDGIGIWDTKSFSIRATKGTKLLVMEVPM EL

In one embodiment, the polynucleotide sequence encoding polypeptide HPhas at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to thepolynucleotide sequence shown as SEQ ID NO 1, SEQ ID NO 3 or SEQ ID NO 5or to variants, homologues, fragments or derivatives thereof.

In one embodiment, the polynucleotide sequence encoding polypeptide HPencodes a polypeptide shown as SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or99% identity to the polypeptide shown as SEQ ID NO 2, SEQ ID NO 4 or SEQID NO 6 or to variants, homologues, fragments or derivatives thereof.

In one embodiment, the polypeptide HP is a truncated HP polypeptide. Forexample, the truncated polypeptide comprises at least 20, 30, 40, 50,75, 100, 125, 150, 175 or 200 amino acids of polypeptide shown as SEQ IDNO 2, SEQ ID NO 4 or SEQ ID NO 6.

In one embodiment, the polynucleotide sequence encoding the polypeptideHP encodes a truncated HP polypeptide.

In one embodiment, the polypeptide HP is a fusion polypeptide. Forexample, the polypeptide is fused to glutathione S-transferase (GST).

Host Cell

In one aspect, a host cell as described herein comprises apolynucleotide sequence encoding polypeptide HP.

In another aspect, a host cell as described herein comprises anexpression vector comprising a polynucleotide sequence encodingpolypeptide HP.

In a further aspect, a host cell as described herein has beentransformed with a nucleotide sequence that causes the host cell tooverexpress HP. For example, a promoter is inserted into the genome of ahost cell which enables the host cell to overexpress a polynucleotidesequence HP (such as an endogenous polynucleotide sequence)—i.e. thepromoter is capable of overexpressing the polynucleotide sequenceencoding HP.

As used herein, the term “overexpress” in the phrase “a nucleotidesequence that causes the host cell to overexpress HP” and “promotercapable of overexpressing the polynucleotide sequence encoding HP”refers to an increase in expression from zero to a level of expressionor going from a lower level of expression to a higher level ofexpression (e.g. upregulation) when the transformed host cell iscompared to the equivalent host cell prior to transformation.

In one embodiment, the level of mRNA encoding HP in a transformed hostcell which overexpresses HP is increased (i.e. upregulated) such thatthe level of mRNA is at least 10%, 20%, 30%, 40% or 50% higher in atransformed host cell when compared to the equivalent host cell prior totransformation.

Examples of host cells overexpressing HP include: (i) host cellstransformed with an expression vector encoding HP (prior totransformation said host cell was not capable of expressing HP); and(ii) host cells transformed to upregulate the expression of anendogenous HP (prior to transformation said host cell was capable ofexpressing said HP for a given set of culture conditions but aftertransformation said host cell is capable of expressing said HP at ahigher level, in the same culture conditions).

The polynucleotide sequence encoding polypeptide HP may be codonoptimised for the host cell. For instance, the polypeptide sequence maybe codon optimised for expression in E. coli (such as SEQ ID NO 3) orthe polynucleotide sequence may be codon optimised for expression inLactococcus lactis (such as SEQ ID NO 5).

The term “host cell”—in relation to the present invention—includes anycell that comprises either the polynucleotide sequence encoding HP asdescribed herein or an expression vector comprising said polynucleotidesequence as described herein. The host cell may be used in therecombinant production of a protein having the specific properties asdefined herein. The host cell may contain a heterologous polynucleotidesequence coding for HP or may be a cell expressing its natural HPpolynucleotide. For example, the host cell may be from Bacteroides sppsuch as Bacteroides thetaiotaomicron.

The term “host cell” as used herein may be interchangably used with“host organism” and “host microorganism”.

Thus, there is provided host cells transformed or transfected with apolynucleotide sequence encoding HP as described herein or an expressionvector comprising said polynucleotide sequence as described herein.

The term “transfected cell” or “transfected host cell” as used hereinmeans a host cell transfected so that it comprises a polynucleotidesequence encoding HP as described herein or an expression vectorcomprising said polynucleotide sequence as described herein. In additionor alternatively, the host cell has been transformed with a nucleotidesequence that causes the host cell to overexpress a polynucleotidesequence encoding HP. For example, a promoter is inserted into thegenome of a host cell which enables the host cell to overexpress anendogenous poly nucleotide sequence encoding HP.

The term “transformed cell” or “transformed host cell” as used hereinmeans a host cell having a modified genetic structure.

The term “host cell” includes any cell which a vector is capable oftransfecting or transducing.

Host cells will be chosen to be compatible with the vector and may forexample be prokaryotic (for example bacterial), fungal, yeast or plantcells.

Host cells comprising polynucleotide sequences encoding polypeptide HPor an expression vector comprising said polynucleotide sequence may beused to express the polypeptide HP under in vitro, in vivo and ex vivoconditions.

In one embodiment, the host cell is a microorganism, such as abacterium. Typically, the microorganism which inhabits the alimentarycanal, or a section of the alimentary canal. Examples of suitablebacterial host cells are gram positive or gram negative bacterialspecies. For instance, the host cell may be selected from the groupconsisting of Bacteroides spp (such as Bacteroides thetaiotaomicron), E.coli, Lactococcus spp (such as L. lactis), Lactobacillus spp,Bifidobacterium spp, and Streptococcus spp (such as Streptococcusthermophilus).

In one embodiment, the host cell comprises an exogenous polynucleotidesequence encoding HP.

In another embodiment, the host cell comprises an endogenouspolynucleotide sequence encoding HP. For example, the endogenouspolynucleotide sequence under the control of a non-native promoter (suchas a constitutive promoter). In a further example, the host cellcomprises multiple copies of the endogenous polynucleotide sequence.

The term “host cell” does not cover native nucleotide coding sequencesin their natural environment when they are under the control of theirnative promoter which is also in its natural environment. An example ofa host cell which has a native nucleotide sequence which is not in itsnatural environment is a Bacteroides thetaiotaomicron VPI-5482comprising SEQ ID NO 1 in which SEQ ID NO 1 under the control of anon-native promoter (such as a constitutive promoter). In anotherexample, a Bacteroides thetaiotaomicron VPI-5482 comprising SEQ ID NO 1has multiple copies of SEQ ID NO 1.

Depending on the nature of the nucleotide sequence, and/or thedesirability for further processing of the expressed protein, eukaryotichosts such as yeasts or other fungi or insect cells (such as insect Sf9cells) may be used. In general, yeast cells are preferred over fungalcells because they are easier to manipulate. However, some proteins areeither poorly secreted from the yeast cell, or in some cases are notprocessed properly (e.g. hyperglycosylation in yeast). In theseinstances, a different fungal host organism should be selected.

The use of suitable host cells—such as yeast, fungal and plant hostcells—may provide for post-translational modifications (e.g.myristoylation, glycosylation, truncation, lapidation and tyrosine,serine or threonine phosphorylation) as may be needed to confer optimalbiological activity on the polypeptide described herein.

Host cells may be cultured under suitable conditions which allowexpression of the polypeptide.

In some embodiments, the polypeptide can be extracted from host cells bya variety of techniques known in the art, including enzymatic, chemicaland/or osmotic lysis and physical disruption. The polypeptide may bepurified and isolated in a manner known per se.

Transformation of Host Cells

Teachings on the transformation of prokaryotic hosts is well documentedin the art, for example see Sambrook et al (Molecular Cloning: ALaboratory Manual, 2nd edition, 1989, Cold Spring Harbor LaboratoryPress). If a prokaryotic host is used then the nucleotide sequence mayneed to be suitably modified before transformation—such as by removal ofintrons.

Filamentous fungi cells may be transformed using various methods knownin the art—such as a process involving protoplast formation andtransformation of the protoplasts followed by regeneration of the cellwall in a manner known. The use of Aspergillus as a host microorganismis described in EP 0 238 023.

Another host organism can be a plant. A review of the general techniquesused for transforming plants may be found in articles by Potrykus (AnnuRev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou(Agro-Food-Industry Hi-Tech Mar./Apr. 1994 17-27). Further teachings onplant transformation may be found in EP-A-0449375.

General teachings on the transformation of fungi, yeasts and plants arepresented in following sections.

Transformed Fungus

A host cell may be a fungus—such as a mould. Examples of suitable suchhosts include any member belonging to the genera Thermomyces,Acremonium, Aspergillus, Penicillium, Mucor, Neurospora, Trichoderma andthe like.

In one embodiment, the host cell may be a filamentous fungus.

Transforming filamentous fungi is discussed in U.S. Pat. No. 5,741,665which states that standard techniques for transformation of filamentousfungi and culturing the fungi are well known in the art. An extensivereview of techniques as applied to N. crassa is found, for example inDavis and de Serres, Methods Enzymol (1971) 17A: 79-143.

Further teachings which may also be utilised in transforming filamentousfungi are reviewed in U.S. Pat. No. 5,674,707.

In addition, gene expression in filamentous fungi is taught in in Puntet al. (2002) Trends Biotechnol 2002 May; 20(5):200-6, Archer & PeberdyCrit Rev Biotechnol (1997) 17(4):273-306.

The present description encompasses the production of transgenicfilamentous fungi according to the present description prepared by useof these standard techniques.

In one aspect, the host organism can be of the genus Aspergillus, suchas Aspergillus niger.

A transgenic Aspergillus according to the present invention can also beprepared by following, for example, the teachings of Turner G. 1994(Vectors for genetic manipulation. In: Martinelli S. D., Kinghorn J. R.(Editors) Aspergillus: 50 years on. Progress in industrial microbiologyvol 29. Elsevier Amsterdam 1994. pp. 641-666).

Transformed Yeast

In another embodiment, the transgenic organism can be a yeast.

A review of the principles of heterologous gene expression in yeast areprovided in, for example, Methods Mol Biol (1995), 49:341-54, and CurrOpin Biotechnol (1997) October; 8(5):554-60

In this regard, yeast—such as the species Saccharomyces cerevisi orPichia pastoris (see FEMS Microbiol Rev (2000 24(1):45-66), may be usedas a vehicle for heterologous gene expression.

A review of the principles of heterologous gene expression inSaccharomyces cerevisiae and secretion of gene products is given by EHinchcliffe E Kenny (1993, “Yeast as a vehicle for the expression ofheterologous genes”, Yeasts, Vol 5, Anthony H Rose and J StuartHarrison, eds, 2nd edition, Academic Press Ltd.).

For the transformation of yeast, several transformation protocols havebeen developed. For example, a transgenic Saccharomyces according to thepresent invention can be prepared by following the teachings of Hinnenet al., (1978, Proceedings of the National Academy of Sciences of theUSA 75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito, Het al (1983, J Bacteriology 153, 163-168).

The transformed yeast cells may be selected using various selectivemarkers—such as auxotrophic markers dominant antibiotic resistancemarkers.

Transformed Plants/Plant Cells

A host cell suitable for the present invention may be a plant. In thisrespect, the basic principle in the construction of genetically modifiedplants is to insert genetic information in the plant genome so as toobtain a stable maintenance of the inserted genetic material. A reviewof the general techniques may be found in articles by Potrykus (Annu RevPlant Physiol Plant Mol Biol [1991] 42:205-225) and Christou(Agro-Food-Industry Hi-Tech Mar./Apr. 1994 17-27).

Direct infection of plant tissues by Agrobacterium is a simple techniquewhich has been widely employed and which is described in Butcher D. N.et al., (1980), Tissue Culture Methods for Plant Pathologists, eds.: D.S. Ingrams and J. P. Helgeson, 203-208.

Other techniques for transforming plants include ballistictransformation, the silicon whisker carbide technique (see Frame B R,Drayton P R, Bagnaall S V, Lewnau C J, Bullock W P, Wilson H M, DunwellJ M, Thompson J A & Wang K (1994) Production of fertile transgenic maizeplants by silicon carbide whisker-mediated transformation, The PlantJournal 6: 941-948) and viral transformation techniques (e.g. see MeyerP, Heidmann I & Niedenhof I (1992) The use of cassava mosaic virus as avector system for plants, Gene 110: 213-217).

Further teachings on plant transformation may be found in EP-A-0449375.

Plant cells may be grown and maintained in accordance with well-knowntissue culturing methods such as by culturing the cells in a suitableculture medium supplied with the necessary growth factors such as aminoacids, plant hormones, vitamins, etc.

In a further aspect, the present description relates to a vector systemwhich carries a nucleotide sequence or construct according to thepresent description and which is capable of introducing the nucleotidesequence or construct into the genome of an organism, such as a plant.The vector system may comprise one vector, but it may comprise twovectors. In the case of two vectors, the vector system is normallyreferred to as a binary vector system. Binary vector systems aredescribed in further detail in Gynheung An et al., (1980), BinaryVectors, Plant Molecular Biology Manual A3, 1-19.

One extensively employed system for transformation of plant cells usesthe Ti plasmid from Agrobacterium tumefaciens or a Ri plasmid fromAgrobacterium rhizogenes An et al., (1986), Plant Physiol. 81, 301-305and Butcher D. N. et al., (1980), Tissue Culture Methods for PlantPathologists, eds.: D. S. Ingrams and J. P. Helgeson, 203-208. Aftereach introduction method of the desired promoter or construct ornucleotide sequence according to the present invention in the plants,the presence and/or insertion of further DNA sequences may be necessary.If, for example, for the transformation the Ti- or Ri-plasmid of theplant cells is used, at least the right boundary and often however theright and the left boundary of the Ti- and Ri-plasmid T-DNA, as flankingareas of the introduced genes, can be connected. The use of T-DNA forthe transformation of plant cells has been intensively studied and isdescribed in EP-A-120516; Hoekema, in: The Binary Plant Vector SystemOffset-drukkerij Kanters B. B., Alblasserdam, 1985, Chapter V; Fraley,et al., Crit. Rev. Plant Sci., 4:1-46; and An et al., EMBO J. (1985)4:277-284.

Culturing and Production

Host cells transformed with the nucleotide sequence descred herein maybe cultured under conditions conducive to the production of the encodedpolypeptide and which facilitate recovery of the polypeptide from thecells and/or culture medium.

The medium used to cultivate the cells may be any conventional mediumsuitable for growing the host cell in questions and obtaining expressionof the polypeptide.

The protein produced by a recombinant cell may be displayed on thesurface of the cell.

The protein may be secreted from the host cells and may conveniently berecovered from the culture medium using well-known procedures.

Secretion

Often, it is desirable for the protein to be secreted from theexpression host cell into the culture medium from where the protein maybe more easily recovered.

According to the present decsription, the secretion leader sequence maybe selected on the basis of the desired expression host. Hybrid signalsequences may also be used with the context of the present invention.

Typical examples of heterologous secretion leader sequences are thoseoriginating from the fungal amyloglucosidase (AG) gene (glaA—both 18 and24 amino acid versions e.g. from Aspergillus), the a-factor gene (yeastse.g. Saccharomyces, Kluyveromyces and Hansenula) or the α-amylase gene(Bacillus).

By way of example, the secretion of heterologous proteins in E. coli isreviewed in Methods Enzymol (1990) 182:132-43.

Expression Vectors

The term “expression vector” means a construct capable of in vivo, exvivo or in vitro expression.

The term “construct”—which is synonymous with terms such as “conjugate”,“cassette” and “hybrid”—includes a nucleotide sequence as describedherein which optionally may be directly or indirectly attached to apromoter. An example of an indirect attachment is the provision of asuitable spacer group such as an intron sequence, such as the Sh1-intronor the ADH intron, intermediate the promoter and the nucleotide sequenceof the present invention. The same is true for the term “fused” inrelation to the present invention which includes direct or indirectattachment. In some cases, the terms do not cover the naturalcombination of the nucleotide sequence coding for the protein ordinarilyassociated with the wild type gene promoter and when they are both intheir natural environment.

The construct may even contain or express a marker, which allows for theselection of the genetic construct.

For some applications, the construct comprises at least the nucleotidesequence described herein operably linked to a promoter.

The nucleotide sequence of the present description may be present in avector in which the nucleotide sequence is operably linked to regulatorysequences capable of providing for the expression of the nucleotidesequence by a suitable host cell.

In some embodiments, the polynucleotide sequence encoding polypeptide HPof an expression vector may be codon optimised for the host cell whichwill be or has been transformed or transfected with the polynucleotidesequence.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A regulatory sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under condition compatible with the controlsequences. For example, a promoter is operably linked to a codingsequence if it controls the transcription of the coding sequence.

The term “regulatory sequences” includes promoters and enhancers andother expression regulation signals.

The term “promoter” is used in the normal sense of the art, e.g. an RNApolymerase binding site. The promoter may be heterologous or homologousto the nucleotide sequence.

Enhanced expression of the nucleotide sequence described herein may alsobe achieved by the selection of heterologous regulatory regions, e.g.promoter, secretion leader and terminator regions.

In one embodiment, the nucleotide sequence as described herein isoperably linked to at least a promoter.

Other promoters may even be used to direct expression of the polypeptidedescribed herein.

Examples of suitable promoters for directing the transcription of thenucleotide sequence in a bacterial, fungal or yeast host are well knownin the art.

The promoter can additionally include features to ensure or to increaseexpression in a suitable host. For example, the features can beconserved regions such as a Pribnow Box or a TATA box.

Once transformed into a suitable host, the vector may replicate andfunction independently of the host genome, or may, in some instances, beincorporated into the genome into the genome of a suitable host cell. Insome instances, the term “incorporated” covers stable incorporation intothe genome.

The vectors for use in the present invention may be transformed into asuitable host cell as described herein to provide for expression of apolypeptide of the present description.

The vector may be a plasmid, a phage particle, or simply a potentialgenomic insert.

The choice of vector e.g. a plasmid, cosmid, or phage vector will oftendepend on the host cell into which it is to be introduced.

The vectors for use in the present invention may contain one or moreselectable marker genes—such as a gene, which confers antibioticresistance e.g. ampicillin, kanamycin, chloramphenicol or tetracyclinresistance. Alternatively, the selection may be accomplished byco-transformation (as described in WO91/17243).

Vectors may be used in vitro, for example for the production of RNA orused to transfect, transform, transduce or infect a host cell.

The vector may further comprise a nucleotide sequence enabling thevector to replicate in the host cell in question. Examples of suchsequences are the origins of replication of plasmids pUC19, pACYC177,pUB110, pE194, pAMB1 and pIJ702.

In one embodiment, an expression vector comprises one or morepolynucleotide sequences according to the present invention. Thepolynucleotide sequence may be heterologous or homologous to a host celltransformed or transfected with the expression vector.

Disorders

Polypeptide HP or a polynucleotide sequence encoding polypeptide HP or ahost cell comprising said polynucleotide sequence, or a host cellcomprising an expression vector comprising said polynucleotide sequencemay be used for the treatment and/or prevention of a disorder in asubject, wherein said disorder is an inflammatory disorder and/or anautoimmune disorder. The disorder may also be of the CNS, includingautism.

In one embodiment, the disorder affects the alimentary canal, a sectionof the alimentary canal, the liver, liver cells, epithelial cells,epidermal cells, neuronal cells, the pancreas, and/or pancreatic cells(such as the islets of Langerhans), kidneys, spleen, lungs and heartand/or cells thereof.

Examples of sections (i.e. parts) of the alimentary canal include themouth, the oesophagus, the stomach and the intestine (such as the smallintestine (e.g. the duodenum, the jejunum and the ileum) and/or thelarge intestine (e.g. the caecum, ascending colon, transverse colon,descending colon, and sigmoid colon)).

Examples of epithelial cells include intestinal, oral, lung, nasal,vaginal epithelial cells.

In one embodiment, the disorder is selected from the group consisting ofinflammatory bowel disorder (IBD), colitis, rheumatoid arthritis,psoriasis, multiple sclerosis, type I diabetes, coeliac disease, atopicdermatitis, rhinitis, irritable bowel syndrome (IBS), ulcerativecolitis, pouchitis, Crohn's disease, functional dyspepsia, atopicdiseases, necrotising enterocolitis, non alcoholic fatty liver disease,gastrointestinal infection, Lupus, nephritis/glomerulonephritis, asthma,COPD, mycocarditis and combinations thereof.

In one aspect, the disorder affects the intestine.

In one aspect, the disorder is an inflammatory disorder. For example,the disorder is an inflammatory bowel disorder (IBD) such as Crohn'sdisease.

In one aspect, the disorder is an autoimmune disorder. For example, theautoimmune disorder is selected from the group consisting of ulcerativecolitis, pouchitis, rheumatoid arthritis, psoriasis, multiple sclerosis,type I diabetes, allergies (including coeliac disease), atopicdermatitis, rhinitis, Lupus, nephritis/glomerulonephritis, asthma, COPDand mycocarditis.

Subject

In one embodiment, the subject is a monogastric animal.

Examples of monogastric animals include poultry, humans, rats, pigs,dogs, cats, horses and rabbits.

In another embodiment, the subject is a mammal such as a monogastricmammal.

Examples of monogastric mammals include omnivores (such as humans, rats,and pigs), carnivores (such as dogs and cats), and herbivores (such ashorses and rabbits).

In one embodiment, the subject is a human.

In one aspect, the subject has a disorder is selected from the groupconsisting of inflammatory bowel disorder (IBD), colitis, rheumatoidarthritis, psoriasis, multiple sclerosis, type I diabetes, coeliacdisease, atopic dermatitis, rhinitis, irritable bowel syndrome (IBS),ulcerative colitis, pouchitis, Crohn's disease, functional dyspepsia,atopic diseases, necrotising enterocolitis, non alcoholic fatty liverdisease, gastrointestinal infection Lupus, nephritis/glomerulonephritis,asthma, COPD, mycocarditis and combinations thereof. For example, thesubject has IBD.

Modulation/Regulation

The terms “modulation” and “regulation” may be used interchangeablyherein.

In one embodiment polypeptide HP, or a polynucleotide sequence encodingpolypeptide HP, or a host cell comprising said polynucleotide sequence,or a host cell comprising an expression vector comprising saidpolynucleotide sequence, is used to modulate the inflammation of a cell,a tissue or an organ in a subject.

In one embodiment, the term “modulation” refers to an increase and/orinduction and/or promotion and/or activation. In an alternativeembodiment, the term “modulation” refers to a decrease and/or reductionand/or inhibition.

In one embodiment, the term “regulation” refers to an upregulation. Inan alternative embodiment, the term “regulation” refers to adownregulation.

In one embodiment, the polypeptide HP or the polynucleotide sequenceencoding polypeptide HP or the host cell as described herein reduces theinflammation of a cell, a tissue or an organ. For example, inflammationof the alimentary canal, a section (i.e. part) of the alimentary canal(such as the intestine), the liver, liver cells, epithelial cells,epidermal cells, neuronal cells, the pancreas, and/or pancreatic cells(such as the islets of Langerhans), kidneys, spleen, lungs and heartand/or cells thereof is reduced.

In one example, inflammation of the alimentary canal or part thereof(such as the intestine) is reduced.

In another example, inflammation by epithelial cells of the tissue orthe organ is reduced.

The term “inflammation” as used herein refers to one or more of thefollowing: redness, swelling, pain, tenderness, heat, and disturbedfunction of a cell, a tissue or organ due to an inflammatory processtriggered by over-reaction of the immune system.

In one embodiment, the numbers of cells which are inflamed in a subjectis at least 10%, 20%, 30%, 40% or 50% lower after administration of thepolypeptide or polynucleotide or host cell as described herein whencompared to the numbers of cells which are inflamed in a subject beforethe polypeptide HP or the polynucleotide sequence encoding HP or a hostcell as described herein is administered to the subject.

In one embodiment, the amount of a tissue or organ which is inflamed ina subject is at least 10%, 20%, 30%, 40% or 50% lower afteradministration of the polypeptide or polynucleotide or host cell asdescribed herein when compared to the amount of tissue or organ which isinflamed in a subject before the polypeptide HP or the polynucleotidesequence encoding HP or a host cell as described herein is administeredto the subject.

In one embodiment, the polypeptide HP or the polynucleotide sequenceencoding polypeptide HP or the host cells as described herein reducesthe inflammation by epithelial cells of the tissue or the organ.

For example, the epithelial cells are epithelial cells of the alimentarycanal or part thereof (such as the intestine).

Without wishing to be bound by theory, the polypeptide HP or thepolynucleotide sequence encoding polypeptide HP or the host cell asdescribed herein increases the production of T cells (such as regulatoryT cells which may also be referred to as Tregs) in a subject. Thisincrease in Treg numbers may combat the effects of other effector Tcells (also referred to as Teffs), such as Th1, Th17 and Th2 which driveinflammation, autoimmunity and allergic/atopic conditions. In Crohn'sdisease and ulcerative colitis the Teff/Treg cell balance is lost.

In one embodiment, the production of T cells in a subject is increasedsuch that there are at least 10%, 20%, 30%, 40% or 50% more T cells, orgreater than 100% more T cells after administration of the polypeptideor polynucleotide or host cell as described herein when compared to thenumber of T cells in the subject before the polypeptide HP or thepolynucleotide sequence encoding HP or the host cell as described hereinis administered to the subject.

Intestine Barrier Integrity

In one embodiment, the polypeptide HP, or a polynucleotide sequenceencoding polypeptide HP, or a host cell comprising said polynucleotidesequence, or a host cell comprising an expression vector comprising saidpolynucleotide sequence, is used to improve intestine barrier integrityin a subject.

The term “improving intestine barrier integrity” as used herein refersto a reduction in the numbers and/or types of microorganisms whichspread from the intestine into other cells in a subject afteradministration of the polypeptide or polynucleotide or host cells asdescribed herein when compared to the numbers and/or types ofmicroorganisms which spread from the intestine into other cells in asubject before administration of the polypeptide or polynucleotide orhost cell as described herein.

In one embodiment, the numbers of microorganisms which spread from theintestine into other cells in a subject are at least 10%, 20%, 30%, 40%or 50% lower after administration of the polypeptide or polynucleotideor host cell as described herein when compared to the numbers ofmicroorganisms which spread from the intestine into other cells in asubject before the polypeptide HP or the polynucleotide sequenceencoding HP or the host cell as described herein is administered to thesubject.

In one embodiment, there are at least 5%, 10%, 15% or 20% fewer types ofmicroorganisms which spread from the intestine into other cells in asubject after administration of the polypeptide or polynucleotide orhost cell as described herein when compared to the types ofmicroorganisms which spread from the intestine into other cells in asubject before the polypeptide HP or the polynucleotide sequenceencoding HP or a host cell as described herein is administered to thesubject.

Levels of Bacteria

In one embodiment polypeptide HP, or a polynucleotide sequence encodingpolypeptide HP, or a host cell comprising said polynucleotide sequence,or a host cell comprising an expression vector comprising saidpolynucleotide sequence, is used to modify the bacterial composition ina tissue or organ to provide a more beneficial microbiota. For example,the invention can be used to reduce the level of one or more types oflactose fermenting bacteria (such as E. coli) in a tissue or an organ ina subject and/or reduce the level of one or more types of non-lactosefermenting bacteria in a tissue or an organ in a subject.

The term “reduce the level of one or more types of lactose fermentingbacteria” as used herein refers to a reduction in the numbers of lactosefermenting bacteria in a tissue or organ in a subject afteradministration of the polypeptide or polynucleotide or host cell asdescribed herein when compared to the numbers of lactose fermentingbacteria in a tissue or organ in a subject before administration of thepolypeptide or polynucleotide or host cell as described herein.

In one embodiment, the numbers of lactose fermenting bacteria in atissue or organ in a subject are at least 10%, 20%, 30%, 40% or 50%lower after administration of the polypeptide or polynucleotide or hostcell as described herein when compared to the numbers of lactosefermenting bacteria in a tissue or organ in a subject before thepolypeptide HP or the polynucleotide sequence encoding HP or the hostcell as described herein is administered to the subject.

Examples of lactose fermenting bacteria include E. coli, Enterobacterand Klebsiella.

The term “reduce the level of one or more types of non-lactosefermenting bacteria” as used herein refers to a reduction in the numbersof non-lactose fermenting bacteria in a tissue or organ in a subjectafter administration of the polypeptide or polynucleotide or host cellswhen compared to the numbers of non-lactose fermenting bacteria in atissue or organ in a subject before administration of the polypeptide orpolynucleotide or host cell as described herein.

In one embodiment, the numbers of non-lactose fermenting bacteria in atissue or organ in a subject are at least 10%, 20%, 30%, 40% or 50%lower after administration of the polypeptide or polynucleotide or hostcell as described herein when compared to the numbers of non-lactosefermenting bacteria in a tissue or organ in a subject before thepolypeptide HP or the polynucleotide sequence encoding HP or the hostcell as described herein is administered to the subject.

Examples of non-lactose fermenting bacteria include as Salmonella,Proteus species, Pseudomonas aeruginosa and Shigella.

In one embodiment the tissue or organ is selected from the groupconsisting of mesenteric lymph nodes, liver, pancreas, spleen andcombinations thereof.

Regulating Appetite and/or Weight

In one embodiment, polypeptide HP or a polynucleotide sequence encodingsaid polypeptide or a host cell comprising said polynucleotide sequence,or a host cell comprising an expression vector comprising saidpolynucleotide sequence is used to regulate the appetite (e.g. foodintake) in a subject (such as a subject with IBD).

As used herein, the term “regulate appetite” or “regulating appetite”refers to the ability to modulate (e.g. increase or decrease) the desirefor a subject to eat food.

In one embodiment, the term “regulate” or “regulation” refers to anincrease in appetite (e.g. food intake). In an alternative embodiment,the term “regulate” or “regulation” refers to a decrease in appetite(e.g. food intake).

For example, the polypeptide or polynucleotide or host cell as describedherein maintains or stimulates the appetite in the subject.

In one embodiment, polypeptide HP or a polynucleotide sequence encodingsaid polypeptide or a host cell comprising said polynucleotide sequence,or a host cell comprising an expression vector comprising saidpolynucleotide sequence is used to regulate the weight of a subject(such as a subject with IBD).

In one embodiment, the term “regulate” or “regulation” refers to anincrease in weight. In an alternative embodiment, the term “regulate” or“regulation” refers to a decrease in weight.

For example, the polypeptide or polynucleotide or host cell as describedherein maintains the weight of a subject or increases the weight of asubject.

Without wishing to be bound by theory, the polypeptide HP or thepolynucleotide sequence encoding HP or the host cell as described hereinexerts a stimulatory effect on the appetite of a subject bydownregulating the expression of genes associated with the suppressionof appetite (such as genes encoding satiety hormones). Agt, Cartpt, Cck,Cxcl12 and Gcg are examples of genes associated with regulating appetiteand the downregulation of one or more of these genes is associated withthe suppression of appetite.

Cholecystokinin (Cck) and glucagon (Gcg) are examples of satietyhormones.

In one aspect, the polypeptide HP or the polynucleotide sequenceencoding HP or the host cell as described herein stimulates the appetitein the subject such that the subject consumes at least 5%, 10%, or 15%more food after administration of the polypeptide or polynucleotide orhost cell as described herein when compared to the subject before thepolypeptide HP or the polynucleotide sequence encoding HP or the hostcell as described herein is administered to the subject. In addition, oralternatively, the polypeptide HP or the polynucleotide sequenceencoding HP or the host cell as described herein stimulates the appetitein the subject such that after 1 month from first administration of thepolypeptide or polynucleotide or host cell as described herein theweight of the subject is at least 2%, 5%, or 10% higher when compared tothe subject before the polypeptide HP or the polynucleotide sequenceencoding HP or the host cell as described herein is administered to thesubject.

In one embodiment, the polypeptide HP or the polynucleotide sequenceencoding HP or the host cell as described herein reduces the level ofcholecystokinin (Cck) and/or glucagon (Gcg) in the blood of a subject.

In one aspect, the polypeptide HP or the polynucleotide sequenceencoding HP or the host cell as described herein reduces the level ofcholecystokinin (Cck) and/or glucagon (Gcg) in the blood of a subject byat least 5%, 10%, 15% or 20% after administration of the polypeptide orpolynucleotide or host cell as described herein when compared to thesubject before the polypeptide HP or the polynucleotide sequenceencoding HP or the host cell as described herein is administered to thesubject.

In one embodiment, the polypeptide HP or the polynucleotide sequenceencoding HP or the host cell as described herein downregulates theexpression of the gene encoding cholecystokinin (Cck) and/or theexpression of the gene encoding glucagon (Gcg) in a cell or cells of asubject.

In one aspect, the polypeptide HP or the polynucleotide sequenceencoding HP or the host cell as described herein decreases theexpression of the gene encoding cholecystokinin (Cck) such that theexpression level (e.g. mRNA level) is at least 5%, 10%, 15% or 20% lowerafter administration of the polypeptide or polynucleotide or host cellas described herein when compared to the expression level in the subjectbefore the polypeptide HP or the polynucleotide sequence encoding HP orthe host cell as described herein is administered to the subject.

In one aspect, the polypeptide HP or the polynucleotide sequenceencoding HP or the host cell as described herein decreases theexpression of the gene encoding glucagon (Gcg) such that the expressionlevel (e.g. mRNA level) is at least 5%, 10%, 15% or 20% lower afteradministration of the polypeptide or polynucleotide or host cell asdescribed herein when compared to the expression level in the subjectbefore the polypeptide HP or the polynucleotide sequence encoding HP orthe host cell as described herein is administered to the subject.

Maintaining the Length of Part of the Intestine

In one embodiment polypeptide HP, or a polynucleotide sequence encodingpolypeptide HP, or a host cell comprising said polynucleotide sequence,or a host cell comprising an expression vector comprising saidpolynucleotide sequence, is used to maintain the length of part of theintestine (such as the large intestine and/or small intestine) of asubject.

Examples of sections (i.e. parts) of the intestine include the smallintestine (e.g. the duodenum, the jejunum and the ileum) and/or thelarge intestine (e.g. the caecum, ascending colon, transverse colon,descending colon, and sigmoid colon).

The term “maintains the length” as used herein refers to there being noor only a small change in the length of part of the intestine afteradministration of the polypeptide or polynucleotide or host cell asdescribed herein when compared to the length of that part of theintestine before administration of the polypeptide or polynucleotide orhost cell as described herein.

In one embodiment, the polypeptide or polynucleotide sequence or hostcell as described herein prevents a reduction in the length of largeintestine. In addition or alternatively, the polypeptide orpolynucleotide sequence or host cell as described herein prevents anincrease in the length of the small intestine.

In one embodiment, a small change in the length of the large intestineof a subject is a reduction in length of less than 5%, 2% or 1% afteradministration of the polypeptide or polynucleotide or host cell asdescribed herein when compared to the length of the large intestine in asubject before the polypeptide HP or the polynucleotide sequenceencoding HP or the host cell as described herein is administered to thesubject.

In one embodiment, a small change in the length of the small intestineof a subject is an increase in length of less than 1%, 2%, 5%, 7% or 10%after administration of the polypeptide or polynucleotide or host cellas described herein when compared to the length of the small intestinein a subject before the polypeptide HP or the polynucleotide sequenceencoding HP or the host cell as described herein is administered to thesubject.

Intestine Disruption

In one embodiment, polypeptide HP, or a polynucleotide sequence encodingpolypeptide HP, or a host cell comprising said polynucleotide sequence,or a host cell comprising an expression vector comprising saidpolynucleotide sequence, is used to reduce disruption to the intestine(e.g. large intestine) of a subject (such as a subject with IBD).

The term “disruption to the intestine of a subject” as used hereinrefers to an affect on the integrity of the mucosal epithelium and/or anaffect on the number of goblet cells in the epithelium and/or an affecton the number of immune cells infiltrating the lamina propria.

In one embodiment, the polypeptide or polynucleotide sequence or hostcell of the description reduces or prevents disruption to the integrityof the mucosal epithelium and/or reduces or prevents a reduction in thenumber of goblet cells in the epithelium and/or reduces or prevents theinfiltration of immune cells into the lamina propria.

In one embodiment, a reduction in disruption to the integrity of themucosal epithelium is a reduction of at least 5%, 10%, 15% or 20% in thenumbers of bacteria crossing from the intestinal lumen into intestinalcells after administration of the polypeptide or polynucleotide or hostcell as described herein when compared to the numbers of bacteriacrossing from the intestinal lumen into intestinal cells in a subjectbefore the polypeptide HP or the polynucleotide sequence encoding HP orthe host cell as described herein is administered to the subject.

In one embodiment, an increase in the number of goblet cells in theepithelium is an increase of at least 2%, 5%, 10%, 15% or 20% in thenumbers of goblet cells in the epithelium of a subject afteradministration of the polypeptide or polynucleotide or host cell asdescribed herein when compared to the number goblet cells in theepithelium of a subject before the polypeptide HP or the polynucleotidesequence encoding HP or the host cell as described herein isadministered to the subject.

In one embodiment, the reduction in the infiltration of immune cellsinto the lamina propria is such that over a fixed time period (such as24 hours) there is a reduction of at least 5%, 10%, 15%, 20% or 30% inthe numbers of immune cells (e.g. T cells) crossing into lamina propriaafter administration of the polypeptide or polynucleotide or host cellas described herein when compared to the numbers of immune cells (e.g. Tcells) crossing from the into lamina propria cells in a subject beforethe polypeptide HP or the polynucleotide sequence encoding HP or thehost cell as described herein is administered to the subject.

Pro-Inflammatory Genes and Barrier Integrity Genes

In one embodiment, polypeptide HP, or a polynucleotide sequence encodingpolypeptide HP, or a host cell comprising said polynucleotide sequence,or a host cell comprising an expression vector comprising saidpolynucleotide sequence, is used to regulate the expression of one ormore pro-inflammatory genes and/or anti-inflammatory genes and/or one ormore barrier integrity genes in a cell or cells of a subject.

In one embodiment, the term “regulate” refers to an upregulation in theexpression of one or more pro-inflammatory genes or anti-inflammatorygenes. In an alternative embodiment, the term “regulate” refers to adownregulation in the expression of one or more pro-inflammatory genesor anti-inflammatory genes.

In one embodiment, the polypeptide HP or the polynucleotide sequenceencoding polypeptide HP or the host cell as described hereindownregulates the expression of one or more pro-inflammatory genes in acell or cells of a subject.

The term “pro-inflammatory gene” as used herein refers to a gene which,when expressed, promotes inflammation. Examples of pro-inflammatorygenes include genes encoding but not limited to IL1-β, IL4, IL5, IL6,IL8, IL12, IL13, IL17, IL21, IL22, IL23, IL27, IFNγ, CCL2, CCL3, CCL5,CCL20, CXCL5, CXCL10, CXCL12, CXCL13, and TNF-α.

In one embodiment, the pro-inflammatory gene is selected from the groupconsisting of IL6, CXCL10, and TNF-α.

In one embodiment, the expression level (e.g. mRNA level) of one or morepro-inflammatory genes is decreased (i.e. downregulated) such that thelevel is at least 10%, 20%, 30%, 40% or 50% lower after administrationof the polypeptide or polynucleotide or host cell as described hereinwhen compared to the level in the subject before polypeptide HP or thepolynucleotide sequence encoding polypeptide HP or the host cell asdescribed herein is administered to the subject.

The term “barrier integrity genes” as used herein refers to a genewhich, when expressed, has a role in the function of the barrier of theintestine such as the repair of the barrier and the prevention ofmicroorganisms crossing the barrier. Examples of barrier integrity genesinclude genes encoding Retnlg|Retnlb, Si, Defa24, Hsd11b2, Hsd17b2, andNr1d1|Thra.

In one embodiment, the term “regulate” refers to an upregulation in theexpression of one or more barrier integrity genes. In an alternativeembodiment, the term “regulate” refers to a downregulation in theexpression of one or more barrier integrity genes.

In one embodiment, the polypeptide HP or the polynucleotide sequenceencoding polypeptide HP or the host cell as described herein upregulatesthe expression of barrier integrity genes in a cell or cells of asubject

In one embodiment, the barrier integrity gene is selected from the groupconsisting of Retnlg|Retnlb, Si, Defa24, Hsd11b2, Hsd17b2, andNr1d1|Thra.

In one embodiment, the expression level (e.g. mRNA level) of one or morebarrier integrity genes is increased (i.e. upregulated) such that thelevel is at least 10%, 20%, 30%, 40% or 50% higher after administrationof the polypeptide or polynucleotide or host cell as described hereinwhen compared to the level in the subject before polypeptide HP or thepolynucleotide sequence encoding polypeptide HP or the host cell asdescribed herein is administered to the subject.

In one embodiment, the polypeptide HP or the polynucleotide sequenceencoding polypeptide HP or the host cell as described herein upregulatesthe expression of anti-inflammatory genes.

Regulating Gene Expression

In one embodiment, the polypeptide HP, or a polynucleotide sequenceencoding polypeptide HP, or a host cell comprising said polynucleotidesequence, or a host cell comprising an expression vector comprising saidpolynucleotide sequence, is used in regulating the expression in a cellor cells of a subject (such as a subject with IBD) of one or more genesselected from the group consisting of regenerating islet-derived 3 betagene (Reg3b), resistin-like gamma resistin like beta gene(Retnlg|Retnlb), sucrase-isomaltase (alpha-glucosidase) gene (Si),defensin alpha 24 gene (Defa24), hydroxysteroid 11-beta dehydrogenase 2gene (Hsd11b2), hydroxysteroid (17-beta) dehydrogenase 2 gene (Hsd17b2),resistin-Like Molecule-beta (RELMb), and nuclear receptor 1D1 thyroidhormone receptor alpha gene (Nr1d1|Thra).

The terms “Reg”, “Reg3” and “Reg3b” as used herein are interchangeable.

The terms “Hsd”, “Hsd17b2” or “Hsd17b2” as used herein areinterchangeable.

In one embodiment, the term “regulate” refers to an upregulation in theexpression of the genes. In an alternative embodiment, the term“regulate” refers to a downregulation in the expression of the genes.

The present invention is useful in regulating the expression ofpro-inflammatory genes and/or barrier integrity genes.

For the avoidance of doubt, pro-inflammatory genes include IL-β, IL4,IL5, IL6, IL8, IL12, IL13, IL17, IL21, IL22, IL23, IL27, IFNα, CCL2,CCL3, CCL5, CCL20, CXCL5, CXCL10, CXCL12, CXCL13 and TNF-α.

For the avoidance of doubt, barrier integrity genes includeRetnlg/Retnlb, Si, Defa24, Hsd11b2, Hsd17b2, and Nrd1\Thra.

In one embodiment, the polypeptide or polynucleotide sequence or hostcell as described herein decreases the expression of one or more genesselected from the group consisting of regenerating islet-derived 3 betagene (Reg3b); resistin-like gamma resistin like beta gene(Retnlg|Retnlb); resistin-Like Molecule-beta (RELMb), sucrase-isomaltase(alpha-glucosidase) gene (Si); and defensin alpha 24 gene (Defa24). Forexample, the expression level (e.g. mRNA level) of one or more genesselected from the group is decreased (i.e. downregulated) such that thelevel is at least 10%, 20%, 30%, 40% or 50% lower after administrationof the polypeptide or polynucleotide or host cell as described hereinwhen compared to the level in the subject before polypeptide HP or thepolynucleotide sequence encoding polypeptide HP or the host cell asdescribed herein is administered to the subject.

In one embodiment, the polypeptide or polynucleotide sequence or hostcell as described herein increases the expression of one or more genesselected from the group consisting of hydroxysteroid 11-betadehydrogenase 2 gene (Hsd11b2); hydroxysteroid (17-beta) dehydrogenase 2gene (Hsd17b2); and nuclear receptor 1D1 thyroid hormone receptor alphagene (Nr1d1|Thra). For example, the expression level (e.g. mRNA level)of one or more genes selected from the group is increased (i.e.upregulated) such that the level is at least 10%, 20%, 30%, 40% or 50%higher after administration of the polypeptide or polynucleotide or hostcell as described herein when compared to the level in the subjectbefore polypeptide HP or the polynucleotide sequence encodingpolypeptide HP or the host cell as described herein is administered tothe subject.

Proinflammatory Pathways

In one embodiment, polypeptide HP or a polynucleotide sequence encodingpolypeptide HP, or a host cell comprising said polynucleotide sequence,or a host cell comprising an expression vector comprising saidpolynucleotide sequence, is used to reduce the activation ofpro-inflammatory pathways in a cell or cells of a subject.

The reduction in the activation of pro-inflammatory pathways can bedetermined by determining the inflammation in a subject.

Inflammation in a subject can be determined by determining the levels ofpro-inflammatory cytokines and chemokines in tissue, serum and/or faecalsamples in a subject before, and after, the polypeptide orpolynucleotide or host cell as described herein is administered to thesubject. For example, the levels of one or more of the following can bemonitored: IL-1, IL-4, IL5, IL6, IL-8, IL-12, IL-13, IL-17, IL-21,IL-22, IL23, TNFα, IFNγ, CXCL1, CXCL10, CCL20 serum and faecalcalprotectin, SA1009/SA1008 calcium binding proteins, and Type 1interferons, CD markers such as CD163, CD14, inflammatory transcriptionfactors such as NF-κβ, STAT, and MAPkinases, c-reactive protein (CRP),erythrocyte sedimentation rate (ESR), complement proteins, serumalbumin, histological evaluation of target tissues and organs, diseaseactivity indices.

In one embodiment, polypeptide HP, or a polynucleotide sequence encodingpolypeptide HP, or a host cell comprising said polynucleotide sequence,or a host cell comprising an expression vector comprising saidpolynucleotide sequence, is used to reduce the activity and/orexpression of NF-κβ in a cell or cells (such as epithelial cells,epidermal cells, neuronal cells, liver, spleen, kidney, lung, heartand/or pancreatic cells) of a subject.

For example, the activity of NF-κβ is decreased such that the activityof NF-κβ is at least 10%, 20%, 30%, 40% or 50% lower afteradministration of the polypeptide or polynucleotide or host cell asdescribed herein when compared to the level in the subject before thepolypeptide HP or the polynucleotide sequence encoding polypeptide HP orthe host cell as described herein is administered to the subject.

For example, the expression level (e.g. mRNA) of NF-κβ is decreased(i.e. downregulated) such that the level is at least 10%, 20%, 30%, 40%or 50% lower after administration of the polypeptide or polynucleotideor host cell as described herein when compared to the level in thesubject before the polypeptide HP or the polynucleotide sequenceencoding polypeptide HP or the host cell as described herein isadministered to the subject.

Alimentary Canal

Parts of the alimentary canal include the mouth, the oesophagus, thestomach and the intestine (such as the small intestine (e.g. theduodenum, the jejunum and the ileum) and/or the large intestine (e.g.the caecum, ascending colon, transverse colon, descending colon, andsigmoid colon).

Herein, the term “large intestine” may be used interchangeably with theterm “colon”.

In one embodiment, polypeptide HP, or a polynucleotide sequence encodingpolypeptide HP, or a host cell comprising said polynucleotide sequence,or a host cell comprising an expression vector comprising saidpolynucleotide sequence, is used for improving alimentary canal healthin a subject.

The term “improving alimentary canal health” as used herein refers toreducing the level of inflammation in the alimentary canal or partthereof and/or improving intestinal microbiota.

In one embodiment, the level of inflammation in the alimentary canal isat least 10%, 20%, 30%, 40% or 50% lower after administration of thepolypeptide or polynucleotide or host cell as described herein whencompared to the level of inflammation in the alimentary canal of asubject before the polypeptide or polynucleotide or host cell asdescribed herein is administered to the subject.

In one embodiment, polypeptide HP, or a polynucleotide sequence encodingpolypeptide HP, or a host cell comprising said polynucleotide sequence,or a host cell comprising an expression vector comprising saidpolynucleotide sequence, is used for improving intestinal microbiota ina subject.

The term “intestinal microbiota” as used herein refers to microorganismsthat live in the digestive tract of the host animals. Thesemicroorganisms perform a wide variety of metabolic, structural,protective and other beneficiary functions.

As used herein, the term “improving intestinal microbiota” refers toincreasing the number and/or type of desirable microorganisms present inthe intestine of a subject (e.g. the host), and/or increasing theactivity of said desirable microorganisms in terms of their metabolic,structural, protective and other beneficiary functions. The term“improving intestinal microbiota” may also refer to decreasing thenumber and/or type of undesirable microorganisms present in theintestine of a subject (e.g. the host), and/or decreasing the activityof said undesirable microorganisms in terms of their metabolic,structural, protective and other beneficiary functions.

Microorganisms which are desirable in the intestine of a host are thosemicroorganisms which have a protective and beneficiary function.Firmicutes and/or Bacteroidetes bacteria are examples of desirablemicroorganisms in the intestine of a host.

Microorganisms which are undesirable in the intestine of a host arethose microorganisms which can interfere with the metabolic, structural,protective and other beneficiary functions of desirable microorganismsin the intestine have a protective and beneficiary function. In additionor alternatively, undesirable microorganisms are those which cause, forexample, inflammation and/or diarrhoea. E. coli (ETEC, EPEC, EIEC, EHECand/or EAEC) is an example of an undesirable microorganism in theintestine of a host.

For example, the numbers (i.e. levels) of Firmicutes and/orBacteroidetes bacteria are increased and the numbers of E. coli arereduced; such an improvement in intestinal microbiota may occur insubjects with inflammatory bowel disease (IBD) once the polypeptide HPor the polynucleotide sequence encoding polypeptide HP or a host cellcomprising said polynucleotide sequence or a host cell comprising anexpression vector comprising said polynucleotide sequence has beenadministered to the subject.

In one embodiment, the number of desirable microorganisms (such asFirmicutes and/or Bacteroidetes bacteria) present in the intestine of asubject (e.g. the host), is increased such that the number ofmicroorganisms is at least 10%, 20%, 30%, 40% or 50% higher, or greaterthan 100% higher after administration of the polypeptide orpolynucleotide or host cell as described herein when compared to thelevel in the subject before the polypeptide or polynucleotide or hostcell as described herein is administered to the subject. In addition, oralternatively, the types of desirable microorganisms (such asClostridium cluster XlVa bacteria) present in the intestine of a subject(e.g. the host), are increased such that there are at least 2%, 5%, 10%,or 15% more types of microorganisms after administration of thepolypeptide or polynucleotide or host cell as described herein whencompared to the types in the subject before the polypeptide orpolynucleotide or host cell as described herein is administered to thesubject.

In one embodiment, the protein of the invention modifies the bacterialcomposition in the intestine of a subject to provide a beneficialmicrobiota. For example, the number of undesirable microorganisms (suchas E. coli (ETEC, EPEC, EIEC, EHEC and/or EAEC)) present in theintestine of a subject (e.g. the host), is decreased such that thenumber of microorganisms is at least 10%, 20%, 30%, 40% or 50% lowerafter administration of the polypeptide or polynucleotide or host cellas described herein when compared to the level in the subject before thepolypeptide or polynucleotide or host cell as described herein isadministered to the subject. In addition, or alternatively, the types ofundesirable microorganisms (such as E. coli) present in the intestine ofa subject (e.g. the host), are decreased such that there are at least1%, 2%, 5%, or 10%, fewer types of undesirable microorganisms afteradministration of the polypeptide or polynucleotide or host cell asdescribed herein when compared to the types in the subject before thepolypeptide or polynucleotide or host cell as described herein isadministered to the subject.

Encapsulation

In one embodiment, the polynucleotide sequence encoding polypeptide HPor a host cell comprising said polynucleotide sequence or a host cellcomprising an expression vector comprising said polynucleotide sequenceis encapsulated.

In a further embodiment, a pharmaceutical composition comprising thepolynucleotide sequence encoding polypeptide HP or a host cellcomprising said polynucleotide sequence or a host cell comprising anexpression vector comprising said polynucleotide sequence isencapsulated.

In another embodiment, a nutritional supplement comprising thepolynucleotide sequence encoding polypeptide HP or a host cellcomprising said polynucleotide sequence or a host cell comprising anexpression vector comprising said polynucleotide sequence encoding saidpolypeptide is encapsulated.

In a further embodiment, a feedstuff, food product, dietary supplement,or food additive as described herein is encapsulated.

The term “encapsulated” as used herein refers to a means for protectingthe polypeptide or polynucleotide or host cell as described herein froman incompatible environment by physical separation so that it can bedelivered to the target site (e.g. the intestine) without degradation orsignificant degradation in order that the polypeptide or polynucleotideor host cell can have an effect on the target site. An example is anenteric coated capsule or an enterically-resistant capsule.

Even when the objective of the encapsulation is the isolation of thepolypeptide or polynucleotide or host cell from its surroundings, theprotective coating or shell must be ruptured at the time of desiredaction. The rupturing of the protective coating or shell is typicallybrought about through the application of chemical and physical stimulisuch as pressure, enzyme attack, chemical reaction and physicaldisintegration.

For example, encapsulation ensures that the polypeptide orpolynucleotide or host cell can be ingested so that the polypeptide orpolynucleotide or host cell can be delivered to the target site (e.g.the intestine) in an amount which is effective to produce an effect atthe target site.

Pharmaceutical Composition

In one embodiment, a pharmaceutical composition comprises polypeptideHP, or a polynucleotide sequence encoding polypeptide HP, or a host cellcomprising said polynucleotide sequence, or a host cell comprising anexpression vector comprising said polynucleotide sequence, andoptionally a pharmaceutically acceptable excipient, carrier or diluent.

The pharmaceutical composition may be any pharmaceutical composition. Inone aspect, the pharmaceutical composition is to be administered orally,enterally or rectally. For example, the composition may be an ediblecomposition. “Edible” means a material that is approved for human oranimal consumption.

The pharmaceutical compositions may be for human or animal usage inhuman and veterinary medicine.

Examples of such suitable excipients for the various different forms ofpharmaceutical compositions described herein may be found in the“Handbook of Pharmaceutical Excipients, 2nd Edition, (1994), Edited by AWade and PJ Weller.

Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).

Examples of suitable carriers include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol and the like.

Examples of suitable diluents include one or more of: water, ethanol,glycerol, propylene glycol and glycerin, and combinations thereof.

The choice of pharmaceutical carrier, excipient or diluent can beselected with regard to the intended route of administration andstandard pharmaceutical practice. The pharmaceutical compositions maycomprise as, or in addition to, the carrier, excipient or diluent anysuitable binder(s), lubricant(s), suspending agent(s), coating agent(s),solubilising agent(s).

Examples of suitable binders include starch, gelatin, natural sugarssuch as glucose, anhydrous lactose, free-flow lactose, beta-lactose,corn sweeteners, natural and synthetic gums, such as acacia, tragacanthor sodium alginate, carboxymethyl cellulose and polyethylene glycol.

Examples of suitable lubricants include sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like.

Preservatives, stabilizers, dyes and even flavouring agents may beprovided in the pharmaceutical composition. Examples of preservativesinclude sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid. Antioxidants and suspending agents may be also used.

In one aspect, the polypeptide or polynucleotide sequence or host cellof the pharmaceutical composition is encapsulated.

In another aspect, the polypeptide of the pharmaceutical composition isa recombinant polypeptide.

In a further aspect, the polynucleotide sequence of the pharmaceuticalcomposition encodes a recombinant polypeptide.

In another aspect, the host cell of the pharmaceutical compositionproduces or is capable of producing a recombinant polypeptide.

In a further aspect, an expression vector comprises said polynucleotidesequence of the pharmaceutical composition.

The pharmaceutical may be in the form of a solution or as asolid—depending on the use and/or the mode of application and/or themode of administration.

As used herein, the term “medicament” encompasses medicaments for bothhuman and animal usage in human and veterinary medicine. In addition,the term “medicament” as used herein means any substance, which providesa therapeutic and/or beneficial effect. The term “medicament” as usedherein is not necessarily limited to substances, which need MarketingApproval, but may include substances which, can be used in cosmetics,nutraceuticals, food (including feeds and beverages for example),probiotic cultures, nutritional supplements and natural remedies. Inaddition, the term “medicament” as used herein encompasses a productdesigned for incorporation in animal feed, for example livestock feedand/or pet food.

Nutritional Supplements

Nutritionally acceptable carriers, diluents and excipients include thosesuitable for human or animal consumption and that are used as standardin the food industry. Typical nutritionally acceptable carriers,diluents and excipients will be familiar to the skilled person in theart.

In one embodiment, a nutritional supplement comprises polypeptide HP, ora polynucleotide sequence encoding polypeptide HP, or a host cellcomprising said polynucleotide sequence, or a host cell comprising anexpression vector comprising said polynucleotide sequence, and anutritional acceptable excipient, carrier or diluent.

In one example, the polypeptide or polynucleotide sequence or host cellof the nutritional supplement is encapsulated.

In another example, the polypeptide of the nutritional supplement is arecombinant polypeptide.

In a further aspect, the polynucleotide sequence of the nutritionalsupplement encodes a recombinant polypeptide.

In another aspect, the host cell of the nutritional supplement producesor is capable of producing a recombinant polypeptide.

In a further example, the polynucleotide of the nutritional supplementis comprised in an expression vector.

Feedstuff/Products

A further aspect of the invention relates to feedstuffs, food products,dietary supplements and food additives comprising polypeptide HP or apolynucleotide sequence encoding polypeptide HP or a host cellcomprising said polynucleotide sequence or a host cell comprising anexpression vector comprising said polynucleotide sequence.

The terms “feedstuff”, “food product” “food additive” and “dietarysupplement” as used herein are intended to cover all consumable productsthat can be solid, jellied or liquid.

The term “food product” is used in a broad sense—and covers food forhumans as well as food for animals (i.e. a feed). In one aspect, thefood product is for human consumption. Examples of food products includediary products (such as milk, cheese, beverages comprising whey protein,milk drinks, lactic acid bacteria drinks, yoghurt, drinking yoghurt),bakery products, beverages and beverage powders.

The “feedstuff”, “food product” “food additive” and “dietary supplement”may be in the form of a solution or as a solid—depending on the useand/or the mode of application and/or the mode of administration.

As used herein the term “dietary supplement” includes a formulationwhich is or can be added to a food product or feedstuff as a nutritionalsupplement. The term “dietary supplement” as used here also refers toformulations which can be used at low levels in a wide variety ofproducts that require gelling, texturising, stabilising, suspending,film-forming and structuring, retention of juiciness and improvedmouthfeel, without adding viscosity.

Suitable food products may include, for example, functional foodproducts, food compositions, pet food, livestock feed, health foods,feedstuffs and the like. In one aspect, the food product is a healthfood.

As used herein, the term “functional food product” means food that iscapable of providing not only a nutritional effect, but is also capableof delivering a further beneficial effect to the consumer. Accordingly,functional foods are ordinary foods that have components or ingredients(such as those described herein) incorporated into them that impart tothe food a specific functional—e.g. medical or physiologicalbenefit—other than a purely nutritional effect.

Examples of specific food products that are applicable to the presentinvention include milk-based products, ready to eat desserts, powdersfor re-constitution with, e.g., milk or water, chocolate milk drinks,malt drinks, ready-to-eat dishes, instant dishes or drinks for humans orfood compositions representing a complete or a partial diet intended forpets or livestock.

In one aspect, the feedstuff, food product, dietary supplement or foodadditive according to the present invention are intended for humans,pets or livestock such as monogastric animals. The feedstuff, foodproduct, dietary supplement or food additive may be intended for animalsselected from the group consisting of dogs, cats, pigs, horses, orpoultry. In a further embodiment, the food product, dietary supplementor food additive is intended for adult species, in particular humanadults.

The term “milk-based product” as used herein means any liquid orsemi-solid milk or whey based product having a varying fat content. Themilk-based product can be, e.g., cow's milk, goat's milk, sheep's milk,skimmed milk, whole milk, milk recombined from powdered milk and wheywithout any processing, or a processed product, such as yoghurt, curdledmilk, curd, sour milk, sour whole milk, butter milk and other sour milkproducts. Another important group includes milk beverages, such as wheybeverages, fermented milks, condensed milks, infant or baby milks;flavoured milks, ice cream; milk-containing food such as sweets.

The feedstuffs, food products, dietary supplements or food additives ofthe present invention may be—or may be added to—food supplements, alsoreferred to herein as dietary or nutritional supplements or foodadditives.

The feedstuffs, food products, dietary supplements or food additivesaccording to the invention may also be used in animal nutrition (e.g. inpig nutrition), particularly in the early-weaned period and growingfattening period. The feedstuffs, food products, dietary supplements orfood additives are expected to enhance immune function reduce andprevent infectious diseases, beneficially alter the microbiotacomposition, and improve growth and performance of animals, for example,through increased feed conversion efficiency.

In one embodiment the feedstuff, food product, dietary supplement, orfood additive is encapsulated.

In one embodiment, the polypeptide of the feedstuff, food product,dietary supplement, or food additive is a recombinant polypeptide.

In one example, the polynucleotide of the feedstuff, food product,dietary supplement, or food additive is comprised in an expressionvector.

Administration

The pharmaceutical compositions, the nutritional supplements,feedstuffs, food products, dietary supplements or food additives of thepresent invention may be adapted for oral, rectal, vaginal, parenteral,intramuscular, intraperitoneal, intraarterial, intrathecal,intrabronchial, subcutaneous, intradermal, intravenous, nasal, buccal orsublingual routes of administration.

In one aspect, the pharmaceutical compositions, the nutritionalsupplements, feedstuffs, food products, dietary supplements or foodadditives of the present invention are adapted for oral, rectal,vaginal, parenteral, nasal, buccal or sublingual routes ofadministration.

In a further aspect, the pharmaceutical compositions, the nutritionalsupplements, feedstuffs, food products, dietary supplements or foodadditives of the present invention are adapted for oral administration.

For oral administration, particular use is made of compressed tablets,pills, tablets, gellules, drops, and capsules.

Other forms of administration comprise solutions or emulsions which maybe injected intravenously, intraarterially, intrathecally,subcutaneously, intradermally, intraperitoneally or intramuscularly, andwhich are prepared from sterile or sterilisable solutions. Thepharmaceutical compositions of the present invention may also be in formof suppositories, pessaries, suspensions, emulsions, lotions, ointments,creams, gels, sprays, solutions or dusting powders.

An alternative means of transdermal administration is by use of a skinpatch. For example, the active ingredient can be incorporated into acream consisting of an aqueous emulsion of polyethylene glycols orliquid paraffin. In another example, the active ingredient can also beincorporated into an ointment consisting of a white wax or white softparaffin base together with such stabilisers and preservatives as may berequired.

Pharmaceutical compositions, the nutritional supplements, feedstuffs,food products, dietary supplements or food additives may be formulatedin unit dosage form, i.e., in the form of discrete portions containing aunit dose, or a multiple or sub-unit of a unit dose.

Dosage

A person of ordinary skill in the art can easily determine anappropriate dose of polypeptide HP or a polynucleotide sequence or ahost cell as described herein to administer to a subject without undueexperimentation. Typically, a physician will determine the actual dosagewhich will be most suitable for an individual patient and it will dependon a variety of factors including the activity of the specific bacterialstrain employed, the metabolic stability and length of action of thatstrain, the age, body weight, general health, sex, diet, mode and timeof administration, rate of excretion, drug combination, the severity ofthe particular condition, and the individual undergoing therapy. Thedosages disclosed herein are exemplary of the average case. There can ofcourse be individual instances where higher or lower dosage ranges aremerited, and such are within the scope of this invention.

Combinations

In one aspect, polypeptide HP or a polynucleotide sequence encodingpolypeptide HP or a host cell comprising said polynucleotide sequence ora host cell comprising an expression vector comprising saidpolynucleotide sequence are administered in combination with one or moreother active agents. In such cases, polypeptide HP or a polynucleotidesequence encoding polypeptide HP or a host cell comprising saidpolynucleotide sequence or a host cell comprising an expression vectorcomprising said polynucleotide sequence may be administeredconsecutively, simultaneously or sequentially with the one or more otheractive agents.

For instance, at least two of the polypeptide HP, the polynucleotidesequence and the host cell as described herein are administered to thesubject.

For example, one type of host cell according the present invention (e.g.a L. lactis transformed with a polynucleotide sequence encoding HP) maybe combined with another type of host cell according to the presentinvention (e.g. a Lactobacillus spp transformed with a polynucleotidesequence encoding HP).

In another example, one type of host cell according the presentinvention (e.g. a L. lactis transformed with a polynucleotide sequenceencoding HP) may be combined with another microorganism such asBacteroides spp (such as Bacteroides thetaiotaomicron), Lactococcus spp(such as L. lactis), Lactobacillus spp, Bifidobacterium spp, andStreptococcus spp (such as Streptococcus thermophilus).

Polynucleotide Sequence

The scope of the present description encompasses polynucleotidesequences encoding HP polypeptides.

The term “nucleotide sequence” as used herein refers to anoligonucleotide sequence or polynucleotide sequence, and variant,homologues, fragments and derivatives thereof (such as portionsthereof). The nucleotide sequence may be of genomic or synthetic orrecombinant origin, which may be double-stranded or single-strandedwhether representing the sense or anti-sense strand.

The term “nucleotide sequence” in relation to the present descriptionincludes genomic DNA, cDNA, synthetic DNA, and RNA. In one embodiment itmeans cDNA sequence.

In one embodiment, the nucleotide sequence when relating to and whenencompassed by the per se scope of the present description does notinclude the native nucleotide sequence when in its natural environmentand when it is linked to its naturally associated sequence(s) thatis/are also in its/their natural environment. For ease of reference,herein this embodiment is called the “non-native nucleotide sequence”.In this regard, the term “native nucleotide sequence” means an entirenucleotide sequence that is in its native environment and whenoperatively linked to an entire promoter with which it is naturallyassociated, which promoter is also in its native environment. However,the amino acid sequence encompassed by scope the present description canbe isolated and/or purified post expression of a nucleotide sequence inits native organism. In one embodiment, however, the amino acid sequenceencompassed by scope of the present description may be expressed by anucleotide sequence in its native organism but wherein the nucleotidesequence is not under the control of the promoter with which it isnaturally associated within that organism.

Typically, the nucleotide sequence encompassed by the scope of thepresent description is prepared using recombinant DNA techniques (i.e.recombinant DNA). However, in an alternative embodiment of theinvention, the nucleotide sequence could be synthesised, in whole or inpart, using chemical methods well known in the art (see Caruthers M H etal., (1980) Nuc Acids Res Symp Ser 215-23 and Horn T et al., (1980) NucAcids Res Symp Ser 225-232).

The polynucleotide encompassed in the present description may be used inconjunction with other polynucleotide sequences. Thus the presentdescription also covers a combination of polynucleotide sequenceswherein the combination comprises the polynucleotide sequence encodingHP and another polynucleotide sequence, which may be anotherpolynucleotide sequence encoding HP.

Preparation of the Nucleotide Sequence

A nucleotide sequence encoding either a peptide of the presentdescription may be identified and/or isolated and/or purified from anycell or organism producing said peptide. Various methods are well knownwithin the art for the identification and/or isolation and/orpurification of nucleotide sequences. By way of example, DNAamplification techniques to prepare more of a sequence may be used oncea suitable sequence has been identified and/or isolated and/or purified.

By way of further example, a genomic DNA and/or cDNA library may beconstructed using chromosomal DNA or messenger RNA from the organismproducing the peptide. If the amino acid sequence is known, labelledoligonucleotide probes may be synthesised and used to identify clonesfrom the genomic library prepared from the organism. Alternatively, alabelled oligonucleotide probe containing sequences homologous to asimilar known gene could be used to identify clones. In the latter case,hybridisation and washing conditions of lower stringency are used.

Alternatively, clones comprising the peptides of the present descriptioncould be identified by inserting fragments of genomic DNA into anexpression vector, such as a plasmid, transforming bacteria with theresulting genomic DNA library, and then plating the transformed bacteriaonto agar plates containing a substrate for the peptide thereby allowingclones expressing the peptide to be identified.

In a yet further alternative, the nucleotide sequence encoding thepeptide may be prepared synthetically by established standard methods,e.g. the phosphoroamidite method described by Beucage S. L. et al.,(1981) Tetrahedron Letters 22, p 1859-1869, or the method described byMatthes et al., (1984) EMBO J. 3, p 801-805. In the phosphoroamiditemethod, oligonucleotides are synthesised, e.g. in an automatic DNAsynthesiser, purified, annealed, ligated and cloned in appropriatevectors.

The nucleotide sequence may be of mixed genomic and synthetic origin,mixed synthetic and cDNA origin, or mixed genomic and cDNA origin,prepared by ligating fragments of synthetic, genomic or cDNA origin (asappropriate) in accordance with standard techniques. Each ligatedfragment corresponds to various parts of the entire nucleotide sequence.The DNA sequence may also be prepared by polymerase chain reaction (PCR)using specific primers, for instance as described in U.S. Pat. No.4,683,202 or in Saiki R K et al., (Science (1988) 239, pp 487-491).

Amino Acid Sequences

The scope of the present description also encompasses HP polypeptides asdefined herein.

The amino acid sequence may be prepared/isolated from a suitable source,or it may be made synthetically or it may be prepared by use ofrecombinant DNA techniques.

The polypeptide encompassed in the present description may be used inconjunction with other peptides. Thus the present description alsocovers a combination of peptides wherein the combination comprises thepolypeptide HP and another peptide, which may be another HP polypeptide.

The amino acid sequence when relating to and when encompassed by the perse scope of the present invention is not a native peptide. In thisregard, the term “native peptide” means an entire peptide that is in itsnative environment and when it has been expressed by its nativenucleotide sequence.

Recombinant Polypeptide

In one aspect the polypeptide sequence for use in the present inventionis a recombinant sequence—i.e. a sequence that has been prepared usingrecombinant DNA techniques (such as the expression of the polypeptideusing a host cell comprising an expression vector encoding thepolypeptide).

These recombinant DNA techniques are within the capabilities of a personof ordinary skill in the art. Such techniques are explained in theliterature, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis,1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3,Cold Spring Harbor Laboratory Press.

Fusion Proteins

The polypeptide sequence for use according to the present invention maybe produced as a fusion protein, for example to aid in extraction andpurification. Examples of fusion protein partners includeglutathione-S-transferase (GST), 6×His, GAL4 (DNA binding and/ortranscriptional activation domains) and (β-galactosidase). It may alsobe convenient to include a proteolytic cleavage site between the fusionprotein partner and the protein sequence of interest to allow removal offusion protein sequences.

Typically, the fusion protein will not hinder the activity of theprotein sequence.

Gene fusion expression systems in E. coli have been reviewed in CurrOpin Biotechnol (1995) 6(5):501-6.

In another embodiment of the description, the polypeptide sequence maybe ligated to a heterologous sequence to encode a fusion protein. Forexample, for screening of peptide libraries for agents capable ofaffecting the substance activity, it may be useful to encode a chimericsubstance expressing a heterologous epitope that is recognised by acommercially available antibody.

Sequence Identity or Sequence Homology

The terms “polypeptide”, “polypeptide sequence”, “peptide”, “protein”and “amino acid sequence” are used interchangeably herein.

The terms “polynucleotide sequence” and “nucleotide sequence” are usedinterchangeably herein.

The present invention also encompasses the use of sequences having adegree of sequence identity or sequence homology with amino acidsequence(s) of a polypeptide described herein (e.g. variants, homologuesand derivatives) or of any nucleotide sequence encoding such apolypeptide (hereinafter referred to as a “homologous sequence(s)”).Here, the term “homologue” means an entity having a certain homologywith the subject amino acid sequences and the subject nucleotidesequences. Here, the term “homology” can be equated with “identity”.

In the present context, a homologous sequence is taken to include anamino acid or a nucleotide sequence which may be at least 50, 60, 70,75, 80, 85 or 90% identical, in some embodiments at least 95, 96, 97, 98or 99% identical to the subject sequence. Although homology can also beconsidered in terms of similarity (i.e. amino acid residues havingsimilar chemical properties/functions), in the context of the presentinvention it is preferred to express homology in terms of sequenceidentity.

In some embodiments, a homologous sequence is taken to include an aminoacid sequence or nucleotide sequence which has one or several additions,deletions and/or substitutions compared with the subject sequence.

In some embodiments, the present invention relates to the use of aprotein whose amino acid sequence is represented herein or a proteinderived from this (parent) protein by substitution, deletion or additionof one or several amino acids, such as 2, 3, 4, 5, 6, 7, 8, 9 aminoacids, or more amino acids, such as 10 or more than 10 amino acids inthe amino acid sequence of the parent protein and having the activity ofthe parent protein.

In some embodiments, the present invention relates to the use of anucleic acid sequence (or gene) encoding a protein whose amino acidsequence is represented herein or encoding a protein derived from this(parent) protein by substitution, deletion or addition of one or severalamino acids, such as 2, 3, 4, 5, 6, 7, 8, 9 amino acids, or more aminoacids, such as 10 or more than 10 amino acids in the amino acid sequenceof the parent protein and having the activity of the parent protein.

In the present context, a homologous sequence is taken to include anucleotide sequence which may be at least 50, 60, 70, 75, 85 or 90%identical, in some embodiments at least 95, 96, 97, 98 or 99% identicalto a nucleotide sequence encoding a polypeptide described herein (thesubject sequence). Typically, the homologues will comprise the same orequivalent sequences that code for the domain(s) etc. as the subjectsequence. Although homology can also be considered in terms ofsimilarity (i.e. amino acid residues having similar chemicalproperties/functions), in the context of the present invention it ispreferred to express homology in terms of sequence identity.

The homologous amino acid sequence and/or nucleotide sequence mayprovide and/or encode a polypeptide which retains the functionalactivity and/or enhances the activity of the polypeptide.

In some aspects, an amino acid sequence as described herein has at least50, 60, 70, 75, 80, 85 or 90% identity, in some embodiments at least 95,96, 97, 98 or 99% identity to the subject sequence.

In some aspects, a nucleotide sequence as described herein has at least50, 60, 70, 75, 80, 85 or 90% identity, in some embodiments at least 95,96, 97, 98 or 99% identity to the subject sequence.

Homology comparisons can be conducted by eye, or more usually, with theaid of readily available sequence comparison programs. Thesecommercially available computer programs can calculate % homologybetween two or more sequences.

% homology may be calculated over contiguous sequences, i.e. onesequence is aligned with the other sequence and each amino acid in onesequence is directly compared with the corresponding amino acid in theother sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues.

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion will cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without penalizing unduly the overall homology score. This isachieved by inserting “gaps” in the sequence alignment to try tomaximize local homology.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment so that, for the same number of identicalamino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimized alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified.Typically the default values are used when using such software forsequence comparisons.

Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the Vector NTI (Invitrogen Corp.). Examples of softwarethat can perform sequence comparisons include, but are not limited to,the BLAST package (see Ausubel et al 1999 Short Protocols in MolecularBiology, 4th Ed—Chapter 18), BLAST 2 (see FEMS Microbiol Lett 1999174(2): 247-50; FEMS Microbiol Lett 1999 177(1): 187-8 andtatiana@ncbi.nlm.nih.gov), FASTA (Altschul et al 1990 J. Mol. Biol.403-410) and AlignX for example. At least BLAST, BLAST 2 and FASTA areavailable for offline and online searching (see Ausubel et al 1999,pages 7-58 to 7-60).

Although the final % homology can be measured in terms of identity, thealignment process itself is typically not based on an all-or-nothingpair comparison. Instead, a scaled similarity score matrix is generallyused that assigns scores to each pairwise comparison based on chemicalsimilarity or evolutionary distance. An example of such a matrixcommonly used is the BLOSUM62 matrix—the default matrix for the BLASTsuite of programs. Vector NTI programs generally use either the publicdefault values or a custom symbol comparison table if supplied (see usermanual for further details). For some applications, it is preferred touse the default values for the Vector NTI package.

Alternatively, percentage homologies may be calculated using themultiple alignment feature in Vector NTI (Invitrogen Corp.), based on analgorithm, analogous to CLUSTAL (Higgins D G & Sharp P M (1988), Gene73(1), 237-244).

Once the software has produced an optimal alignment, it is possible tocalculate homology, for example % sequence identity. The softwaretypically does this as part of the sequence comparison and generates anumerical result.

Should Gap Penalties be used when determining sequence identity, thenthe following parameters can be used for pairwise alignment for example:

FOR BLAST GAP OPEN 0 GAP EXTENSION 0

FOR CLUSTAL DNA PROTEIN WORD SIZE  2  1 K triple GAP PENALTY 15 10 GAPEXTENSION  6.66  0.1

In one embodiment, CLUSTAL may be used with the gap penalty and gapextension set as defined above.

In one embodiment, the degree of identity with regard to a nucleotidesequence is determined over at least 20 contiguous nucleotides, forexample over at least 30 contiguous nucleotides, for example over atleast 40 contiguous nucleotides, for example over at least 50 contiguousnucleotides, for example over at least 60 contiguous nucleotides, forexample over at least 100 contiguous nucleotides, for example over atleast 200 contiguous nucleotides, for example over at least 300contiguous nucleotides.

In one embodiment, the degree of identity with regard to a nucleotidesequence may be determined over the whole sequence.

The sequences may also have deletions, insertions or substitutions ofamino acid residues which produce a silent change and result in afunctionally equivalent substance. Deliberate amino acid substitutionsmay be made on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues as long as the secondary binding activity of the substance isretained. For example, negatively charged amino acids include asparticacid and glutamic acid; positively charged amino acids include lysineand arginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine, valine,glycine, alanine, asparagine, glutamine, serine, threonine,phenylalanine, and tyrosine.

Conservative substitutions may be made, for example according to theTable below. Amino acids in the same block in the second column andpreferably in the same line in the third column may be substituted foreach other:

ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M N Q Polar -charged D E K R AROMATIC H F W Y

The present invention also encompasses homologous substitution(substitution and replacement are both used herein to mean theinterchange of an existing amino acid residue, with an alternativeresidue) that may occur i.e. like-for-like substitution such as basicfor basic, acidic for acidic, polar for polar etc. Non-homologoussubstitution may also occur i.e. from one class of residue to another oralternatively involving the inclusion of unnatural amino acids such asornithine (hereinafter referred to as Z), diaminobutyric acid ornithine(hereinafter referred to as B), norleucine ornithine (hereinafterreferred to as O), pyriylalanine, thienylalanine, naphthylalanine andphenylglycine.

Replacements may also be made by unnatural amino acids include; alpha*and alpha-disubstituted* amino acids, N-alkyl amino acids*, lacticacid*, halide derivatives of natural amino acids such astrifluorotyrosine*, p-Cl-phenylalanine*, p-Br-phenylalanine*,p-l-phenylalanine*, L-allyl-glycine*, β-alanine*, L-α-amino butyricacid*, L-γ-amino butyric acid*, L-α-amino isobutyric acid*, L-ε-aminocaproic acid^(#), 7-amino heptanoic acid*, L-methionine sulfone^(#)*,L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine*,L-hydroxyproline^(#), L-thioproline*, methyl derivatives ofphenylalanine (Phe) such as 4-methyl-Phe*, pentamethyl-Phe*, L-Phe(4-amino)^(#), L-Tyr (methyl)*, L-Phe (4-isopropyl)*, L-Tic(1,2,3,4-tetrahydroisoquinoline-3-carboxyl acid)*, L-diaminopropionicacid^(#) and L-Phe (4-benzyl)*. The notation * has been utilised for thepurpose of the discussion above (relating to homologous ornon-homologous substitution), to indicate the hydrophobic nature of thederivative whereas # has been utilised to indicate the hydrophilicnature of the derivative, #* indicates amphipathic characteristics.

Variant amino acid sequences may include suitable spacer groups that maybe inserted between any two amino acid residues of the sequenceincluding alkyl groups such as methyl, ethyl or propyl groups inaddition to amino acid spacers such as glycine or β-alanine residues. Afurther form of variation, involves the presence of one or more aminoacid residues in peptoid form, will be well understood by those skilledin the art. For the avoidance of doubt, “the peptoid form” is used torefer to variant amino acid residues wherein the α-carbon substituentgroup is on the residue's nitrogen atom rather than the α-carbon.Processes for preparing peptides in the peptoid form are known in theart, for example Simon R J et al., PNAS (1992) 89(20), 9367-9371 andHorwell D C, Trends Biotechnol. (1995) 13(4), 132-134.

The nucleotide sequences for use in the present invention may includewithin them synthetic or modified nucleotides. A number of differenttypes of modification to oligonucleotides are known in the art. Theseinclude methylphosphonate and phosphorothioate backbones and/or theaddition of acridine or polylysine chains at the 3′ and/or 5′ ends ofthe molecule. For the purposes of the present invention, it is to beunderstood that the nucleotide sequences described herein may bemodified by any method available in the art. Such modifications may becarried out in order to enhance the in vivo activity or life span ofnucleotide sequences of the present invention.

The present invention also encompasses the use of nucleotide sequencesthat are complementary to the sequences presented herein, or anyderivative or fragment thereof. If the sequence is complementary to afragment thereof then that sequence can be used as a probe to identifysimilar coding sequences in other organisms etc.

Polynucleotides which are not 100% homologous to the sequences of thepresent invention but fall within the scope of the invention can beobtained in a number of ways. Other variants of the sequences describedherein may be obtained for example by probing DNA libraries made from arange of individuals, for example individuals from differentpopulations. In addition, other homologues may be obtained and suchhomologues and fragments thereof in general will be capable ofselectively hybridising to the sequences shown in the sequence listingherein. Such sequences may be obtained by probing cDNA libraries madefrom or genomic DNA libraries from other animal species, and probingsuch libraries with probes comprising all or part of any one of thesequences in the attached sequence listings under conditions of mediumto high stringency. Similar considerations apply to obtaining specieshomologues and allelic variants of the polypeptide or nucleotidesequences of the invention.

Variants and strain/species homologues may also be obtained usingdegenerate PCR which will use primers designed to target sequenceswithin the variants and homologues encoding conserved amino acidsequences within the sequences of the present invention. Conservedsequences can be predicted, for example, by aligning the amino acidsequences from several variants/homologues. Sequence alignments can beperformed using computer software known in the art. For example the GCGWisconsin PileUp program is widely used.

The primers used in degenerate PCR will contain one or more degeneratepositions and will be used at stringency conditions lower than thoseused for cloning sequences with single sequence primers against knownsequences.

Alternatively, such polynucleotides may be obtained by site directedmutagenesis of characterised sequences. This may be useful where forexample silent codon sequence changes are required to optimise codonpreferences for a particular host cell in which the polynucleotidesequences are being expressed. Other sequence changes may be desired inorder to introduce restriction enzyme recognition sites, or to alter theproperty or function of the polypeptides encoded by the polynucleotides.

Polynucleotides (nucleotide sequences) of the invention may be used toproduce a primer, e.g. a PCR primer, a primer for an alternativeamplification reaction, a probe e.g. labelled with a revealing label byconventional means using radioactive or non-radioactive labels, or thepolynucleotides may be cloned into vectors. Such primers, probes andother fragments will be at least 15, preferably at least 20, for exampleat least 25, 30 or 40 nucleotides in length, and are also encompassed bythe term polynucleotides of the invention as used herein.

Polynucleotides such as DNA polynucleotides and probes according to theinvention may be produced recombinantly, synthetically, or by any meansavailable to those of skill in the art. They may also be cloned bystandard techniques.

In general, primers will be produced by synthetic means, involving astepwise manufacture of the desired nucleic acid sequence one nucleotideat a time. Techniques for accomplishing this using automated techniquesare readily available in the art.

Longer polynucleotides will generally be produced using recombinantmeans, for example using a PCR (polymerase chain reaction) cloningtechniques. The primers may be designed to contain suitable restrictionenzyme recognition sites so that the amplified DNA can be cloned into asuitable cloning vector.

Recombinant Polynucleotide Sequence

In one aspect the polynucleotide sequence for use in the presentinvention is a recombinant polypeptide sequence—i.e. a sequence that hasbeen prepared using recombinant DNA techniques (such as the expressionof the polypeptide using a host cell comprising an expression vectorencoding the polypeptide). Examples of recombinant polynucleotidesequences include codon optimised sequences and polynucleotide sequencesencoding fusion polypeptide.

These recombinant DNA techniques are within the capabilities of a personof ordinary skill in the art. Such techniques are explained in theliterature, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis,1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3,Cold Spring Harbor Laboratory Press.

Synthetic

In one aspect the sequence for use in the present invention is asynthetic sequence—i.e. a sequence that has been prepared by in vitrochemical or enzymatic synthesis. It includes, but is not limited to,sequences made with optimal codon usage for host organisms—such as themethylotrophic yeasts Pichia and Hansenula.

The present invention is further described by way of the followingnon-limiting examples.

Examples

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA and immunology, which are within thecapabilities of a person of ordinary skill in the art. Such techniquesare explained in the literature. See, for example, J. Sambrook, E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual,Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel,F. M. et al. (1995 and periodic supplements; Current Protocols inMolecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York,N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation andSequencing: Essential Techniques, John Wiley & Sons; J. M. Polak andJames O′D. McGee, 1990, In Situ Hybridization: Principles and Practice;Oxford University Press; M. J. Gait (Editor), 1984, OligonucleotideSynthesis: A Practical Approach, Irl Press; D. M. J. Lilley and J. E.Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesisand Physical Analysis of DNA Methods in Enzymology, Academic Press; andE. M. Shevach and W. Strober, 1992 and periodic supplements, CurrentProtocols in Immunology, John Wiley & Sons, New York, N.Y. Each of thesegeneral texts is herein incorporated by reference.

Bacteroides thetaiotaomicron HP (BT0187, a pirin-related protein) wasshown in an NF-κβ luciferase reporter assay, to greatly reduce NF-κβactivity stimulated in epithelial cells in culture by flagellin-, PMA-or IL-1.

For large scale production, HP (referred to as HP in FIGS. 1A and 1Bwith FIG. 1A showing the polynucleotide sequence and FIG. 1B showing thepolypeptide sequence) was expressed in E. coli or L. lactis.

The sequence was codon optimised for expression in (i) E. coli (referredto as Rec 1 HP in FIG. 1B) and (ii) L. lactis (referred to as Rec 2 HPin FIG. 1B).

Isolated recombinant HP was tested in vitro and encapsulated.

The efficacy of the encapsulated product was evaluated in a rat model ofinflammatory bowel disease (Dextran Sodium Sulphate [DSS]-inducedcolitis).

Example 1—the Effect of HP on Inflammatory Bowel Disease

Rat study: Hooded-Lister rats (Rowett strain; 6 month-old; ˜480 g) werereared, housed and managed under standard high quality conditions withinthe Bioresources of the Rowett Institute of Nutrition and Health. Theyhad free access to sterile distilled water containing Dextran sodiumsulphate (MP Biomedicals UK, Cambridge; DSS; 36000-50000 mol. wt.) for 7days [days 1-5, 40 g DSS/I and days 6-7, 20 g DSS/I]. Half of theDSS-treated rats were dosed daily (day 1-7) with HP protein and theremaining six DSS-treated rats were dosed daily (day 1-7) with placebo.Untreated controls had free access to sterile distilled water but werenot dosed. Untreated controls had access to sterile distilled water. Allrats had free access to high quality rodent chow. Food intake, waterintake and body weight were measured daily.

The rats were euthanased (isoflurane overdose and exsanguination) anddissected on day 8. The total length of the colon was measured and apiece of ascending colon 3-6 cm from the caecal/colon junction wascollected in OCT or fixed in neutral buffered formalin, 2-3 cm from thecaecal/colon junction was placed in RNAlater and a piece 0-2 cm from thecaecal/colon junction was snap frozen. A piece of descending colon 3-6cm from the rectum was collected in OCT or fixed in neutral bufferedformalin, 2-3 cm from the rectum was placed in RNAlater and a piece 0-2cm from the rectum was snap frozen. The small intestine was measured, apiece of ileal tissue 5-7 cm from the ileocaecal junction was collectedin OCT or fixed in neutral buffered formalin, 7-9 cm from the ileocaecaljunction was collected for microbiology, 9-10 cm from the ileocaecaljunction was placed in RNAlater and a piece 9-17 cm from the ileocaecaljunction was snap frozen. Transverse colon was collected formicrobiology as were mesenteric lymph nodes, liver and spleen.Lactose-fermenting and non-lactose fermenting bacteria in tissues wereevaluated using MacConkey no 3 agar.

Fixed colon samples were embedded in 8100 Technovit resin. 4 μm sectionswere cut and stained with hematoxylin and eosin. Whole transversecross-sectional areas were imaged and digitised using a Zeiss Axioskopmicroscope connected to a Qlmaging camera controlled by ImageProPlussoftware. These were examined in a blinded manner by 2 independentindividuals and the severity of intestinal damage was graded based onthe method of Berg et al. (1996) and data expressed as percentage offields of view with pathology of 0 [no pathology] through to grade 3[major pathology].

The rats treated with Dextran Sodium Sulphate (DSS) and rats treatedwith both DSS and HP (DSS/HP) had comparable water intake and so, theintake of DSS was also same between the treatment groups. At the sametime, it was observed that the food intake by DSS rats was at a slightlylower rate than that of DSS/HP treated rats and controls. The DSStreated rats also tended to lose weight, while at the same time, theDSS/HP and control rats maintained their weight (FIG. 2).

Rat colon length was reduced due to intake of DSS, a reported feature ofDSS colitis. However, this change in the colon was prevented when ratswere also treated with HP (FIG. 3). In contrast, small intestine lengthwas increased with intake of DSS but unaltered in rats given DSS/HP(FIG. 3).

Mesenteric lymph nodes, liver and spleen of rats given DSS weresub-clinically infected with lactose (predominantly E. coli)-fermentingand non-lactose-fermenting bacteria (FIG. 4). This was not evident withDSS/HP. Spread of bacteria to these systemic tissues is likely to beresult of loss of gut barrier integrity due to damage caused by DSS. HPappeared to prevent this loss of gut barrier integrity.

Histological analysis of ascending and descending colon was carried out(FIGS. 5 & 6). The severity of intestinal damage was graded based on themethod of Berg et al. (1996) and data expressed as percentage of fieldsof view with pathology of 0 [no pathology] through to grade 3 [majorpathology] or as mean histopathology score. Disruption to the tissuecaused by DSS was moderate and patchy, with varying degrees of damage(from little or none to severe) localised throughout the tissuesections. Overall, the integrity of the mucosal epithelium was impaired,there was a reduced number of goblet cells in the epithelium andinfiltration of immune cells into the lamina propria. In contrast,overall disruption to the colon caused by DSS was greatly reduced byco-treatment of rats with HP. It was therefore protective in theDSS-induced colitis model.

Affymetrix Analysis

Principal component analysis (PCA) was performed on the microarray datato separate the samples in a 3-dimensional way. Control and DSS/HPclustered together, and DSS were separate from this cluster. This PCAtherefore indicated that DSS transcriptome profile was very differentfrom the control and DSS/HP profiles, which were very similar. In turn,this indicated that HP was effective in treating the inflammation, asthese animals seemed similar to healthy animals.

ANOVA with unequal variance (Welch), P<0.05, asymptotic, all againstsingle condition, Tukey HSD post-hoc was carried out to get a list ofdifferentially expressed genes. This generated a table of 377 genes withdifferential expression (Table 1 and Table 3).

TABLE 1 Genes with differential expression between DSS and Control andDSS/HP and control in rats given Dextran Sodium Sulphate in water withor without co-treatment with hypothetical protein (HP). Fold changeDSS/HP Gene DSS vs vs Gene description symbol Control Control p-valueRegenerating Reg3b 11.400 2.107 0.016 islet-derived 3 beta Resistin-likegamma | Retnlg| 3.957 1.556 0.020 resistin like beta RetnlbSucrase-isomaltase Si 3.903 1.347 0.040 (alpha-glucosidase) Defensin,alpha, 24 Defa24 3.045 1.552 0.026 Hydroxysteroid Hsd11b2 −2.002 1.0010.041 11-beta dehydrogenase 2 Hydroxysteroid Hsd17b2 −2.530 −1.603 0.040(17-beta) dehydrogenase 2 Nuclear receptor Nr3c2 −1.447 −1.092 0.009subfamily 3, group C, member 2 Gene description GO_biological_process(up to first 10) Regenerating islet- GO: 0006953 acute-phase response;derived 3 beta GO: 0006954 inflammatory response Resistin-like gamma |resistin like beta Sucrase-isomaltase GO: 0005975 carbohydrate metabolicprocess; (alpha-glucosidase) GO: 0007568 aging; GO: 0007584 response tonutrient; GO: 0008152 metabolic process; GO: 0009744 response to sucrosestimulus; GO: 0009750 response to fructose stimulus; GO: 0032868response to insulin stimulus; GO:0033189 response to vitamin A; GO:0042594 response to starvation; GO: 0051384 response to glucocorticoidstimulus Defensin, alpha, 24 GO: 0006952 defense response; GO: 0042742defense response to bacterium Hydroxysteroid GO: 0001666 response tohypoxia; GO: 11-beta 0002017 regulation of blood volume by renaldehydrogenase 2 aldosterone; GO: 0006950 response to stress; GO: 0007565female pregnancy; GO: 0008152 metabolic process; GO: 0008211glucocorticoid metabolic process; GO: 0032094 response to food; GO:0032868 response to insulin stimulus; GO: 0042493 response to drug; GO:0048545 response to steroid hormone stimulus Hydroxysteroid GO: 0006694steroid biosynthetic process; (17-beta) GO: 0032526 response to retinoicacid; GO: dehydrogenase 2 0055114 Nuclear receptor GO: 0006355regulation of transcription, DNA- subfamily 1, group D, dependent; GO:0007623 circadian rhythm; member 1 | thyroid GO: 0001502 cartilagecondensation; GO: hormone receptor 0001503 ossification; GO: 0001822kidney alpha development; GO: 0001889 liver development; GO: 0002155regulation of thyroid hormone mediated signaling pathway; GO: 0006950response to stress; GO: 0007420 brain development; GO: 0007611 learningor memory

A heatmap was created of the subset of 377 genes (FIG. 7). This heatmapshowed clear clustering of the DSS animals away from the Control andDSS/HP animals, which themselves also clustered separately, albeit notas distant from each other. The overall colour pattern of control andDSS/HP was very similar, while the DSS animals mostly showed inversefold changes for this gene subset to the other two groups.

Seven of these genes showed comparatively high fold changes compared tocontrols. Of these seven genes, 4 were up regulated and the other 3 weredown regulated with respect to control (Table 1). These fold changeswere generally higher for the DSS animals than for the DSS/HP animals.The most affected genes were regenerating islet-derived 3 beta (Reg3b),resistin-like gamma Iresistin like beta (Retnlg|Retnlb),sucrase-isomaltase (alpha-glucosidase) (Si) and defensin alpha 24(Defa24), which were up regulated and hydroxysteroid 11-betadehydrogenase 2 (Hsd11b2), hydroxysteroid (17-beta) dehydrogenase 2(Hsd17b2), and nuclear receptor 1D1| thyroid hormone receptor alpha(Nr1d1|Thra), which were down regulated with respect to the control(Table 1).

Realtime PCR

Expression of inflammation-associated genes in the ascending colon wasgenerally lower in rats treated with DSS and HP than in tissue from ratstreated with DSS alone (FIG. 8; Table 2). Reg3 and RELMb expression werein particular greatly reduced as a result of treatment with HP.

TABLE 2 Statistical analysis of inflammation-associated genes (RealtimePCR) in ascending colons from rats given Dextran Sodium Sulphate inwater with or without co-treatment with hypothetical protein (HP). DSSvs Control DSS/HP vs Control DSS vs DSS/HP Fold Fold Fold change p-valuechange p-value change p-value RELM-b 6.61 0.03 1.2 0.17 5.49 0.04 Reg361.25 0.04 4.04 0.24 15.15 0.01 Defa24 23.08 0.01 6.37 0.01 3.62 0.22CXCL10 1.1 0.91 1.78 0.48 −1.63 0.39 TNF −1.16 0.9 2.19 0.54 −2.55 0.11Hsd −3.17 0.01 −2.28 0.01 −1.39 0.36 IL6 3.2 0.43 1.35 0.81 2.37 0.11

SUMMARY

Hypothetical protein ameliorated moderate DSS-induced colitis. Thisprotection was, in part, linked with reduced expression ofpro-inflammatory markers in the gut tissue.

TABLE 3 details all genes with differential expression between DSS andControl and DSS/HP and control in rats given Dextran Sodium Sulphate inwater with or without co-treatment with hypothetical protein (HP). Foldchange DSS DSS/HP vs vs p- Gene description Gene symbol Control Controlvalue GO_biological_process (up to first 10) Regenerating islet-derivedReg3b 11.400 2.107 0.016 GO: 0006953 acute-phase response; GO: 0006954 3beta inflammatmy response Resistin-like gamma | Retnlg| 3.957 1.5560.020 resistin like beta Retnlb Sucrase-isomaltase Si 3.903 1.347 0.040GO: 0005975 carbohydrate metabolic process; GO: 0007568(alpha-glucosidase) aging; GO: 0007584 response to nutrient; GO: 0008152metabolic process; GO: 0009744 response to sucrose stimulus; GO: 0009750response to fructose stimulus; GO: 0032868 response to insulin stimulus;GO: 0033189 response to vitamin A; GO: 0042594 response to starvation;GO: 0051384 response to glucocorticoid stimulus Defensin, alpha, 24Defa24 3.045 1.552 0.026 GO: 0006952 defense response GO: 0042742defense response to bacterium Matrix Gla protein Mgp 2.223 1.176 0.048GO: 0001503 ossification; GO: 0006461 protein complex assembly; GO:0007275 multicellular organismal development; GO: 0007584 response tonutrient; GO: 0009612 response to mechanical stimulus; GO: 0009725response to hormone stimulus; GO: 0030154 cell differentiation; GO:0030324 lung development; GO: 0030500 regulation of bone mineralization;GO: 0042221 response to chemical stimulus Phospholipase A2, groupPla2g2a 2.006 1.262 0.027 GO: 0006644 phospholipid metabolic process;GO: 0008285 IIA (platelets, synovial negative regulation of cellproliferation; GO: 0016042 lipid fluid) catabolic process; GO: 0035019somatic stem cell maintenance; GO: 0042127 regulation of cellproliferation; GO: 0046473 phosphatidic acid metabolic process; GO:0050678 regulation of epithelial cell proliferation; GO: 0050680negative regulation of epithelial cell proliferation Gremlin 1, cysteineknot Grem1 1.982 1.338 0.010 GO: 0001658 branching involved in uretericbud superfamily, homolog morphogenesis; GO: 0002689 negative regulationof leukocyte (Xenopus laevis) chemotaxis; GO: 0006915 apoptosis; GO:0007267 cell-cell signaling; GO: 0009887 organ morphogenesis; GO:0009954 proximal/distal pattern formation; GO: 0010717 regulation ofepithelial to mesenchymal transition; GO: 0030308 negative regulation ofcell growth; GO: 0030326 emrnyonic limb morphogenesis; GO: 0030514negative regulation of BMP signaling pathway Ribosomal protein Rpl10a|1.958 1.086 0.033 GO: 0006396 RNA processing. GO: 0006412 translation;L10A | similar to RGD1559639| GO: 0006414 translational elongationribosomal protein L10a RGD1566137 Hydroxysteroid 11-beta Hsd11b1 1.9071.233 0.000 GO: 0006278 RNA-dependent DNA replication; GO: 0006694dehydrogenase 1 steroid biosynthetic process; GO: 0006704 glucocorticoidbiosynthetic process; GO: 0006713 glucocorticoid catabolic process; GO:0008152 metabolic process GO: 0030324 lung development; GO: 0043456regulation of pentose-phosphate shunt Carbamoyl-phosphate Cps1 1.8581.245 0.020 GO: 0000050 urea cycle; GO: 0005980 glycogen catabolicsynthetase 1 process; GO: 0006541 glutamine metabolic process; GO:0006807 nitrogen compound metabolic process; GO: 0014075 response toamine stimulus; GO: 0019433 triglyceride catabolic process; GO: 0032496response to lipopolysaccharide; GO: 0033762 response to glucagonstimulus; GO: 0034201 response to oleic acid. GO: 0042493 response todrug Paraoxonase 3 Pon3 1.816 1.358 0.033 GO: 0019439 aromatic compoundcatabolic process; GO: 0046395 carboxylic acid catabolic processOrnithine Otc 1.816 1.190 0.032 GO: 0000050 urea cycle; GO: 0006526arginine biosynthetic carbamoyltransferase process; GO: 0006591ornithine metabolic process; GO: 0008652 cellular amino acidbiosynthetic process; GO: 0051259 protein oligomerization; GO: 0055081anion homeostasis Leukocyte immunoglobulin- Lilrb4 1.769 1.185 0.013like receptor, subfamily B, member 4 Cadherin 19, type 2 Cdh19 1.7321.328 0.001 GO: 0007155 cell adhesion; GO: 0007156 homophilic celladhesion Complement factor H Cfh 1.723 1.509 0.031 GO: 0006956complement activation GO: 0030449 regulation of complement activationVomeronasal 1 receptor, V1re14 1.715 1.369 0.004 GO: 0007186 G-proteincoupled receptor protein signaling E14 pathway Solute carrier family 25Slc25a4 1.701 1.228 0.010 GO: 0015866 ADP transport; GO: 0015867 ATPtransport; (mitochondrial carrier; GO: 0051935 glutamate uptake involvedin synaptic adenine nucleotide transmission; GO: 0055085 transmembranetransport; GO: translocator), member 4 0060547 negative regulation ofnecrotic cell death Immediate early response Ier3 1.692 1.227 0.037 3ST3 beta-galactoside St3gal4 1.666 −1.069 0.047 GO: 0006486 proteinamino acid glycosylation alpha-2,3-sialyltransferase 4 Fc fragment ofIgG, low Fcgr2a|Fcgr2b| 1.652 1.150 0.013 GO: 0001788 antibody-dependentcellular cytotoxicity; GO: affinity IIa, receptor LOC498276| 0001798positive regulation of type IIa hypersensitivity; GO: (CD32) | Fcfragment of LOC100362543 0001805 positive regulation of type IIIhypersensitivity; GO: IgG, low affinity IIb, 0001812 positive regulationof type I hypersensitivity; GO: receptor (CD32) | Fc gamma 0001820serotonin secretion; GO: 0006910 phagocytosis, receptor II beta | Lowaffinity recognition; GO: 0006911 phagocytosis, engulfment; GO:immunoglobulin gamma Fc 0007166 cell surface receptor linked signalingpathway; region receptor III-like GO: 0021675 nerve development; GO:0030593 neutrophil chemotaxis Eukaryotic translation Eif3e 1.647 1.2870.025 GO: 0000184 nuclear-transcribed mRNA catabolic process, initiationfactor 3, subunit E nonsense-mediated decay; GO: 0006413 translationalinitiation Family with sequence Fam96a 1.641 1.277 0.014 GO: 0008150biological_process similarity 96, member A Peroxisomal membmne Pxmp31.631 1.093 0.007 GO: 0001764 neuron migration; GO: 0006699 bile acidprotein 3 biosynthetic process GO: 0007031 peroxisome organization; GO:0007399 nervous system development; GO: 0008150 biological_process; GO:0042632 cholesterol homeostasis; GO: 0045540 regulation of cholesterolbiosynthetic process; GO: 0001764 neuron migration; GO: 0006699 bileacid biosynthetic process; GO: 0007031 peroxisome organizationFibroblast growth factor Fgf15 1.618 1.045 0.005 GO: 0001755 neuralcrest cell migration; GO: 0007507 heart 15 development; GO: 0008284positive regulation of cell proliferation; GO: 0008543 fibroblast growthfactor receptor signaling pathway; GO: 0046326 positive regulation ofglucose import; GO: 0046330 positive regulation of JNK cascade; GO:0070374 positive regulation of ERK1 and ERK2 cascade; GO: 0070858negative regulation of bile acid biosynthetic process Phospholamban Pln1.616 1.194 0.021 GO: 0002026 regulation of the force of heartcontraction; GO: 0006816 calcium ion transport // non-traceable authorstatement; GO: 0006874 cellular calcium ion homeostasis; GO: 0045822negative regulation of heart contraction; GO: 0048738 cardiac muscletissue development; GO: 0051924 regulation of calcium ion transportSuppressor of cytokine Socs3 1.613 1.157 0.007 GO: 0001558 regulation ofcell growth; GO: 0001666 response signaling 3 to hypoxia; GO: 0001932regulation of protein amino acid phosphorylation; GO: 0007165 signaltransduction; GO: 0007243 intracellular protein kinase cascade; GO:0007259 JAK-STAT cascade; GO: 0007568 aging; GO: 0009408 response toheat; GO: 0009617 response to bacterium; GO: 0009725 response to hormonestimulus PQ loop repeat containing 3 Pqlc3 1.608 1.073 0.037 Midkine Mdk1.600 1.032 0.045 GO: 0000087 M phase of mitotic cell cycle; GO: 0007275multicellular organismal development; GO: 0009611 response to wounding;GO: 0009725 response to hormone stimulus; GO: 0016477 cell migration;GO: 0030154 cell differentiation; GO: 0030325 adrenal gland development;GO: 0042493 response to drug; GO: 0051384 response to glucocorticoidstimulus; GO: 0051781 positive regulation of cell division MOB1, Mps OneBinder Mobkl3 1.593 1.310 0.047 GO: 0006810 transport kinaseactivator-like 3 (yeast) Nuclear factor, interleukin 3 Nfil3 1.589 1.5790.041 GO: 0006355 regulation of transcription, DNA-dependent; regulatedGO: 0048511 rhythmic process Alpha-2u globulin PGCL1 | LOC259246| 1.586−1.100 0.021 GO: 0006810 transport alpha-2u-globulin (L type) |LOC298116| alpha-2u globulin PGCL2 | LOC298109| alpha2u globulin |alpha-2u LOC298111| globulin PGCL3 | alpha 2U LOC259244| globulinLOC366380 Tp53rk binding protein Tprkb 1.580 1.187 0.018 Similar toprotein C33A12.3 RGD1359508 1.574 1.141 0.013 V-ral simian leukemiaviral Rala 1.563 1.175 0.005 GO: 0000910 cytokinesis; GO: 0007165 signaltransduction; oncogene homolog A GO: 0007264 small GTPase mediatedsignal transduction; (ras related) GO: 0007265 Ras protein signaltransduction; GO: 0017157 regulation of exocytosis; GO: 0031532 actincytoskeleton reorganization; GO: 0051491 positive regulation offilopodium assembly; GO: 0051665 membrane raft localizationHematopoietic prostaglandin Hpgds 1.539 1.220 0.009 GO: 0001516prostaglandin biosynthetic process GO: 0006633 D synthase fatty acidbiosynthetic process; GO: 0006693 prostaglandin metabolic processForkhead box E3 Foxe3 1.519 1.131 0.024 GO: 0001654 eye development; GO:0006350 transcription; GO: 0006355 regulation of transcription,DNA-dependent; GO: 0006366 transcription from RNA polymerase IIpromoter; GO: 0008150 biological_process; GO: 0045449 regulation oftranscription; GO: 0048468 cell development; GO: 0050679 positiveregulation of epithelial cell proliferation Complement component 1, C1s1.513 1.170 0.006 GO: 0006508 proteolysis; GO: 0006958 complement ssubcomponent activation, classical pathway; GO: 0010001 glial celldifferentiation; GO: 0045087 innate immune response; GO: 0051591response to cAMP Reticulocalbin 2, EF-hand Rcn2 1.512 1.223 0.035calcium binding domain Solute carrier family 7 Slc7a9 1.500 1.047 0.046GO: 0006865 amino acid transport; GO: 0055085 (cationic amino acidtransmembrane transport; GO: 0015804 neutral amino acid transporter, y+system), transport member 9 Butyrylcholinesterase Bche 1.488 1.042 0.019GO: 0007584 response to nutrient; GO: 0007612 learning; GO: 0019695choline metabolic process; GO: 0042493 response to drug; GO: 0043279response to alkaloid; GO: 0050805 negative regulation of synaptictransmission; GO: 0051384 response to glucocorticoid stimulus; GO:0051593 response to folic acid SFT2 domain containing 1 Sft2d1 1.4871.125 0.023 GO: 0015031 protein transport; GO: 0016192 vesicle-mediatedtransport TCF3 (E2A) fusion partner Tfpt 1.475 1.165 0.002 GO: 0006917induction of apoptosis UDP-Gal: betaGlcNAc beta B4galt4 1.466 1.1880.008 GO: 0005975 carbohydrate metabolic process1,4-galactosyltransferase, polypeptide 4 Somatostatin Sst 1.460 1.1390.020 GO: 0001101 response to acid; GO: 0006972 hyperosmotic response;GO: 0007186 G-protein coupled receptor protein signaling pathway; GO:0009408 response to heat; GO: 0010243 response to organic nitrogen; GO:0030334 regulation of cell migration; GO: 0042493 response to drug; GO:0043200 response to amino acid stimulus; GO: 0048545 response to steroidhormone stimulus Lin-7 homolog C Lin7c 1.459 1.195 0.005 GO: 0006887exocytosis; GO: 0007269 neurotransmitter (C. elegans) secretionGlycoprotein Gpnmb 1.452 1.100 0.036 GO: 0001649 osteoblastdifferentiation GO: 0007155 cell (transmembrane) nmb adhesion GO:0030282 bone mineralization Coiled-coil-helix-coiled- Chchd4| 1.4491.121 0.033 GO: 0015031 protein transport; GO: 0055085 transmembranecoil-helix domain LOC685505 transport containing 4 | similar tocoiled-coil-helix-coiled- coil-helix domain containing 4 Olfactolyreceptor 63 Olr63 1.445 1.147 0.029 GO: 0007165 signal transduction; GO:0007186 G-protein coupled receptor protein signaling pathway; GO:0050911 detection of chemical stimulus involved in sensory perception ofsmell Proline-rich acidic protein 1 Prap1 1.444 1.055 0.035Immunoglobulin superfamily, Igsf6 1.443 1.108 0.045 member 6 Ly49inhibitory receptor 9 | Ly49i9| 1.440 1.026 0.034 hypothetical proteinLOC497796| LOC497796 | killer cell Klra17| lectin-like receptor,RGD1561306 subfamily A, member 17 | similar to immunoreceptor Ly49si3Allograft inflammatmy Aif1 1.431 1.107 0.010 GO: 0001934 positiveregulation of protein amino acid factor 1 phosphorylation; GO: 0010629negative regulation of gene expression; GO: 0014739 positive regulationof muscle hyperplasia; GO: 0030335 positive regulation of cellmigration; GO: 0031668 cellular response to extracellular stimulus; GO:0032870 cellular response to hormone stimulus; GO: 0034097 response tocytokine stimulus; GO: 0042116 macrophage activation; GO: 0043066negative regulation of apoptosis; GO: 0045429 positive regulation ofnitric oxide biosynthetic process Legumain Lgmn 1.427 1.268 0.003 GO:0006508 proteolysis; GO: 0040015 negative regulation of multicellularorganism growth Brain expressed X-linked 2 | Bex2|Bex1| 1.420 1.1940.043 GO: 0006915 apoptosis; GO: 0007049 cell cycle; GO: brain expressedgene 1 | Bex 4 0002052 positive regulation of neuroblast brain expressedgene 4 proliferation; GO: 0007275 multicellular organismal development;GO: 0007399 nervous system development; GO: 0030154 celldifferentiation; GO: 0045665 negative regulation of neurondifferentiation; GO: 0048011 nerve growth factor receptor signalingpathway Tumor suppressor Tusc3 1.419 1.145 0.049 GO: 0045454 cell redoxhomeostasis candidate 3 ATP synthase, H+ Atp5h|Atp5h|1 1.415 1.072 0.036GO: 0006811 ion transport; GO: 0015986 ATP synthesis transporting,mitochondrial coupled proton transport; GO: 0015992 proton transport;GO: F0 complex, subunit d | ATP 0046034 ATP metabolic process synthase,H+ transporting, mitochondrial F0 complex, subunit d-like 1 C-typelectin domain family Clec10a 1.413 −1.051 0.034 10, member A Sodiumchannel, voltage- Scn7a 1.412 1.274 0.017 GO: 0006811 ion transport; GO:0006814 sodium ion gated, type VII, alpha transport; GO: 0055085transmembrane transport Cd55 1.412 1.065 0.015 GO: 0007204 elevation ofcytosolic calcium ion concentration Galactokinase 2 Galk2 1.409 1.1820.000 GO: 0006012 galactose metabolic process GO: 0008152 metabolicprocess GO: 0046835 carbohydrate phosphorylation Necdin homolog (mouse)Ndn 1.402 1.174 0.004 GO: 0001764 neuron migration; GO: 0006355regulation of transcription, DNA-dependent; GO: 0007409 axonogenesis;GO: 0007413 axonal fasciculation; GO: 0007417 central nervous systemdevelopment; GO: 0007585 respiratory gaseous exchange; GO: 0008347 glialcell migration; GO: 0019233 sensory perception of pain; GO: 0048011nerve growth factor receptor signaling pathway; GO: 0048666 neurondevelopment BUD31 homolog Bud31|Ptcd1 1.401 1.098 0.047 (S. cerevisiae)| pentatricopeptide repeat domain 1 Proenkephalin Penk 1.394 1.073 0.017GO: 0001662 behavioral fear response; GO: 0007186 G-protein coupledreceptor protein signaling pathway; GO: 0007218 neuropeptide signalingpathway; GO: 0007610 behavior; GO: 0019233 sensory perception of painMusculoskeletal, embryonic Mustn1 1.394 1.074 0.031 GO: 0030326embryonic limb morphogenesis; GO: 0042060 nuclear protein 1 woundhealing GO: 0042246 tissue regeneration EGF-like module containing, Emr41.380 1.175 0.025 GO: 0007218 neuropeptide signaling pathway mucin-like,hormone receptor- like sequence 4 Testis specific X-linked gene Tsx1.378 1.245 0.000 Lecithin-retinol acyltransferase Lrat 1.369 1.0580.004 GO: 0006776 vitamin A metabolic process; GO: 0007601(phosphatidylcholine-retinol- visual perception; GO: 0009790 embryonicdevelopment; GO: O-acyltransferase) 0042572 retinol metabolic process;GO: 0050896 response to stimulus Integrin, alpha 1 Itga1 1.366 1.0890.013 GO: 0000187 activation of MAPK activity; GO: 0006936 musclecontraction; GO: 0007155 cell adhesion; GO: 0007229 integrin-mediatedsignaling pathway; GO: 0030593 neutrophil chemotaxis; GO: 0042311vasodilation; GO: 0043525 positive regulation of neuron apoptosis; GO:0045123 cellular extravasation; GO: 0048812 neuron projectionmorphogenesis; GO: 0060326 cell chemotaxis Stathmin-like 3 Stmn3 1.3551.164 0.005 GO: 0007019 microtubule depolymerization; GO: 0031122cytoplasmic microtubule organization; GO: 0031175 neuron projectiondevelopment; GO: 0032314 regulation of Rac GTPase activity; GO: 0035021negative regulation of Rac protein signal transduction; GO: 0051493regulation of cytoskeleton organization Family with sequence Fam12b1.344 1.134 0.003 similarity 12, member B (epididymal) Mitochondrialribosomal Mrpl13 1.344 1.089 0.022 GO: 0006412 translation protein L13Similar to RIKEN cDNA RGD1305457 1.344 1.078 0.000 1700023M03 Growthdifferentiation Gdf9 1.343 1.019 0.029 GO: 0001555 oocyte growth; GO:0030308 negative regulation factor 9 of cell growth Olfactory receptor826 | Olr826| 1.334 1.264 0.001 GO: 0007186 G-protein coupled receptorprotein signaling olfactory receptor 825 | Olr825| pathway; GO: 0050911detection of chemical stimulus olfactoly receptor 829 Olr829 involved insensory perception of smell; GO: 0007165 signal transduction Oxidizedlow density Olrl 1.333 1.081 0.022 GO: 0006954 inflammatoly response;GO: 0006955 immune lipoprotein (lectin-like) response; GO: 0007155 celladhesion; GO: 0007159 leukocyte receptor 1 cell-cell adhesion; GO:0008219 cell death; GO: 0042157 lipoprotein metabolic process; GO:0042542 response to hydrogen peroxide Heat shock protein alpha 2 Hspa21.325 1.044 0.023 GO: 0006950 response to stress; GO: 0007275multicellular organismal development; GO: 0007283 spermatogenesis; GO:0030154 cell differentiation Membrane-spanning Ms4a2 1.321 1.129 0.031GO: 0006954 inflammatory response; GO: 0007165 signal 4-domains,subfamily A, transduction; GO: 0007166 cell surface receptor linkedsignaling pathway; GO: 0007202 activation of phospholipase member 2 (Fcfragment C activity; GO: 0007205 activation of protein kinase C activityof IgE, high affinity I, by G-protein coupled receptor protein signalingpathway; receptor for; beta polypeptide) GO: 0043306 positive regulationof mast cell degranulation; GO: 0050663 cytokine secretion; GO: 0051279regulation of release of sequestered calcium ion into cytosol Mesenchymehomeobox 2 Meox2 1.320 1.087 0.013 GO: 0001525 angiogenesis; GO: 0001757somite specification; GO: 0006355 regulation of transcription,DNA-dependent; GO: 0007275 multicellular organismal development; GO:0007519 skeletal muscle tissue development GO: 0060021 palatedevelopment; GO: 0060173 limb development Angiopoietin-like 3 Angptl31.319 1.166 0.002 GO: 0006071 glycerol metabolic process; GO: 0006631fatty acid metabolic process; GO: 0006644 phospholipid metabolicprocess; GO: 0007160 cell-matrix adhesion; GO: 0007165 signaltransduction; GO: 0008203 cholesterol metabolic process; GO: 0009395phospholipid catabolic process; GO: 0009725 response to hormonestimulus; GO: 0010519 negative regulation of phospholipase activity; GO:0019915 lipid storage Phosphotriesterase related Pter 1.318 1.091 0.008GO: 0009056 catabolic process G protein-coupled receptor Gpr119 1.3171.164 0.036 GO: 0007165 signal transduction; GO: 0007186 G-protein 119coupled receptor protein signaling pathway; GO: 0030073 insulinsecretion Similar to 14-3-3 protein LOC298795| 1.309 1.084 0.036 GO:0000079 regulation of cyclin-dependent protein kinase sigma | stratifinSfn activity; GO: 0001836 release of cytochrome c from mitochondria; GO:0008285 negative regulation of cell proliferation; GO: 0008630 DNAdamage response, signal transduction resulting in induction ofapoptosis; GO: 0030216 keratinocyte differentiation; GO: 0030307positive regulation of cell growth; GO: 0043154 negative regulation ofcaspase activity; GO: 0043588 skin development; GO: 0043616 keratinocyteproliferation; GO: 0000079 regulation of cyclin- dependent proteinkinase activity Serpine1 mRNA binding Serbp1 1.307 1.136 0.041 GO:0045767 regulation of anti-apoptosis protein 1 Granzyme F Gzmf 1.3001.043 0.027 GO: 0006508 proteolysis Tissue factor pathway Tfpi 1.2981.198 0.000 GO: 0007596 blood coagulation; GO: 0007598 blood inhibitor(lipoprotein- coagulation, extrinsic pathway associated coagulationinhibitor) Fibroblast growth factor 3 Fgf3 1.294 1.138 0.036 GO: 0001759induction of an organ; GO: 0008284 positive regulation of cellproliferation; GO: 0008543 fibroblast growth factor receptor signalingpathway; GO: 0048538 thymus development Secreted and transmembraneSectm1a 1.292 1.096 0.003 GO: 0043123 positive regulation of I-kappaBkinase/NF- 1A kappaB cascade Ubiquitin-conjugating RGD69425 1.288 1.0590.040 GO: 0008150 biological_process; GO: 0043687 post- enzymetranslational protein modification; GO: 0051246 regulation of proteinmetabolic process Neuropeptide W Npw 1.287 1.009 0.029 GO: 0007186G-protein coupled receptor protein signaling pathway GO: 0007218neuropeptide signaling pathway; GO: 0007631 feeding behavior LOC3625261.282 1.026 0.014 Olfactory receptor 1075 Olr1075 1.279 −1.179 0.027 GO:0007186 G-protein coupled receptor protein signaling pathway; GO:0050911 detection of chemical stimulus involved in sensory perception ofsmell Myelin protein zero-like 1 Mpzl1 1.278 1.269 0.001 Keratin 18Krt18 1.276 1.124 0.048 GO: 0006915 apoptosis; GO: 0008150biological_process; GO: 0033209 tumor necrosis factor-mediated signalingpathway; GO: 0043000 Golgi to plasma membrane CFTR protein transport;GO: 0043066 negative regulation of apoptosis Choline phosphotransferase1 Chpt1 1.273 1.198 0.033 GO: 0006656 phosphatidylcholine biosyntheticprocess; GO: 0006663 platelet activating factor biosynthetic process;GO: 0008654 phospholipid biosynthetic process Neurogenic differentiation1 Neurod1 1.271 1.152 0.020 GO: 0003326 pancreatic A cell fatecommitment; GO: 0003329 pancreatic PP cell fate commitment; GO: 0006355regulation of transcription, DNA-dependent; GO: 0007263 nitric oxidemediated signal transduction; GO: 0007275 multicellular organismaldevelopment; GO: 0007399 nervous system development; GO: 0009749response to glucose stimulus; GO: 0009952 anterior/posterior patternformation; GO: 0021549 cerebellum development; GO: 0030073 insulinsecretion Fibroblast growth factor 2 Fgf2 1.264 1.032 0.042 GO: 0000186activation of MAPKK activity; GO: 0000189 nuclear translocation of MAPK;GO: 0001525 angiogenesis; GO: 0001658 branching involved in ureteric budmorphogenesis; GO: 0001759 induction of an organ; GO: 0001934 positiveregulation of protein amino acid phosphorylation; GO: 0002042 cellmigration involved in sprouting angiogenesis; GO: 0006355 regulation oftranscription, DNA-dependent; GO: 0006700 C21-steroid hormonebiosynthetic process; GO: 0006915 apoptosis Fin bud initiation factorFibin 1.264 1.014 0.001 homolog (zebrafish) Olfactory receptor 7 Olr71.261 1.228 0.011 GO: 0007186 G-protein coupled receptor proteinsignaling pathway; GO: 0050911 detection of chemical stimulus involvedin sensory perception of smell Sterol O-acyltransferase 2 Soat2 1.256−1.037 0.016 GO: 0007584 response to nutrient; GO: 0008202 steroidmetabolic process; GO: 0008203 cholesterol metabolic process; GO:0033344 cholesterol efflux; GO: 0034379 very- low-density lipoproteinparticle assembly; GO: 0034435 cholesterol esterification Neurexin 1Nrxn1 1.256 1.071 0.022 GO: 0007268 synaptic transmission; GO: 0007269neurotransmitter secretion; GO: 0007416 synapse assembly; GO: 0051290protein heterotetramerization MARCKS-like 1 Marcksl1 1.251 1.104 0.033GO: 0008284 positive regulation of cell proliferation GO: 0016192vesicle-mediated transport Calcium/calmodulin- Camk2n1 1.251 1.151 0.024GO: 0007268 synaptic transmission dependent protein kinase II inhibitor1 Armadillo repeat containing, Armcx1 1.247 1.042 0.003 X-linked 1Protocadherin beta 2 Pcdhb2 1.244 1.090 0.037 GO: 0007155 cell adhesionGO: 0007156 homophilic cell adhesion ELAV (embryonic lethal, Elavl21.242 −1.022 0.041 abnormal vision, Drosophila)-like 2 (Hu antigen B)DNA-damage regulated Dram2| 1.239 1.088 0.013 GO: 0006915 apoptosis. GO:0006917 induction of apoptosis autophagy modulator 2 | LOC689412 similarto CG4025-PA Proteolipid protein 1 Plp1 1.238 1.183 0.019 GO: 0007229integrin-mediated signaling pathway; GO: 0008366 axon ensheathment; GO:0010001 glial cell differentiation; GO: 0022010 myelination in thecentral nervous system; GO: 0042552 myelination GO: 0042759 long-chainfatty acid biosynthetic process; GO: 0048469 cell maturationAspartoacylase Aspa 1.237 1.012 0.036 GO: 0008152 metabolic process; GO:0022010 myelination in the central nervous system; GO: 0048714 positiveregulation of oligodendrocyte differentiation First gene upstream ofLOC362863 1.232 1.198 0.037 Nt5dc3 PRKC, apoptosis, WT1, Pawr 1.2321.140 0.049 GO: 0006915 apoptosis; GO: 0030889 negative regulation ofregulator B cell proliferation; GO: 0042094 interleukin-2 biosyntheticprocess; GO: 0042130 negative regulation of T cell proliferation; GO:0042986 positive regulation of amyloid precursor protein biosyntheticprocess; GO: 0045449 regulation of transcription; GO: 0050860 negativeregulation of T cell receptor signaling pathway Glutamate receptor,Gria4 1.231 1.181 0.036 GO: 0007268 synaptic transmission ionotropic,AMPA4 Fumarylacetoacetate Fahd1 1.229 1.152 0.039 GO: 0008152 metabolicprocess hydrolase domain containing 1 McKusick-Kaufman Mkks 1.225 1.1780.010 GO: 0007286 spermatid development; GO: 0007608 sensory syndromeperception of smell GO: 0008150 biological_process; GO: 0009296flagellum assembly; GO: 0021756 striatum development; GO: 0021766hippocampus development; GO: 0021987 cerebral cortex development; GO:0035058 sensory cilium assembly; GO: 0035176 social behavior; GO:0042384 cilium assembly Protein kinase inhibitor, Pkig 1.223 1.226 0.017GO: 0000122 negative regulation of transcription from RNA gammapolymerase II promoter; GO: 0006469 negative regulation of proteinkinase activity; GO: 0007165 signal transduction; GO: 0042308 negativeregulation of protein import into nucleus Cholecystokinin B receptorCckbr 1.222 1.103 0.036 GO: 0001821 histamine secretion; GO: 0002209behavioral defense response; GO: 0006915 apoptosis; GO: 0007165 signaltransduction; GO: 0007186 G-protein coupled receptor protein signalingpathway; GO: 0007204 elevation of cytosolic calcium ion concentration;GO: 0007586 digestion; GO: 0008284 positive regulation of cellproliferation; GO: 0032230 positive regulation of synaptic transmission,GABAergic; GO: 0032868 response to insulin stimulus RAS-like family 11Rasl11b 1.221 1.156 0.028 GO: 0007165 signal transduction GO: 0007264small GTPase member B mediated signal transduction Galanin receptor 1Galr1 1.219 1.117 0.047 GO: 0007165 signal transduction GO: 0007186G-protein coupled receptor protein signaling pathway; GO: 0007189activation of adenylate cyclase activity by G-protein signaling pathwayPotassium voltage gated Kcnb1 1.217 1.040 0.038 GO: 0006811 iontransport; GO: 0006813 potassium ion channel, Shab-related transport;GO: 0051259 protein oligomerization; GO: subfamily, member 1 0055085transmembrane transport Protein disulfide isomerase Pdia4 1.216 1.1590.037 GO: 0045454 cell redox homeostasis family A, member 4 Myosin,light polypeptide 1 Myl1 1.215 1.128 0.024 GO: 0060048 cardiac musclecontraction Persephin Pspn 1.213 1.255 0.006 GO: 0001658 branchinginvolved in ureteric bud morphogenesis Tumor necrosis factor Tnfsf131.212 1.228 0.001 GO: 0002426 immunoglobulin production in mucosaltissue; (ligand) superfamily, GO: 0002636 positive regulation ofgerminal center formation; member 13 GO: 0006955 immune response; GO:0008150 biological_process; GO: 0008284 positive regulation of cellproliferation; GO: 0016064 immunoglobulin mediated immune response; GO:0048298 positive regulation of isotype switching to IgA isotypes; GO:0050776 regulation of immune response ADP-ribosylation factor Arfip11.211 1.039 0.027 GO: 0006886 intracellular protein transport; GO:0050708 interacting protein 1 regulation of protein secretion Zincfinger, MYND-type Zmynd19 1.211 1.043 0.035 containing 19 Centrosomalprotein 70 kDa Cep70 1.210 1.158 0.024 Ribosomal L24 domain Rsl24d11.210 1.139 0.035 GO: 0006412 translation; GO: 0042254 ribosomebiogenesis containing 1 Ring finger protein 133 Rnf133 1.209 1.157 0.018GO: 0051865 protein autoubiquitination Plasma glutamate Pgcp 1.205 1.1470.044 GO: 0006508 proteolysis; GO: 0042246 tissue regenerationcathoxypeptidase DnaJ (Hsp40) homolog, Dnaja1 1.199 1.320 0.014 GO:0006457 protein folding; GO: 0007283 spermatogenesis; subfamily A,member 1 GO: 0009408 response to heat; GO: 0030317 sperm motility; GO:0030521 androgen receptor signaling pathway; GO: 0042769 DNA damageresponse, detection of DNA damage Transmembrane and coiled- Tmco1 1.1981.057 0.011 GO: 0008150 biological_process coil domains 1 Arrestin, beta1 Arrb1 1.196 1.204 0.002 GO: 0000187 activation of MAPK activity; GO:0002031 G- protein coupled receptor internalization; GO: 0002032desensitization of G-protein coupled receptor protein signaling pathwayby arrestin; GO: 0006366 transcription from RNA polymerase II promoter;GO: 0006892 post-Golgi vesicle- mediated transport; GO: 0006897endocytosis; GO: 0007165 signal transduction; GO: 0007186 G-proteincoupled receptor protein signaling pathway; GO: 0007188 G-proteinsignaling, coupled to cAMP nucleotide second messenger //; GO: 0007600sensory perception Dystonia 1 Dyt1 1.193 1.043 0.002 GO: 0006457 proteinfolding; GO: 0006979 response to oxidative stress; GO: 0051085 chaperonemediated protein folding requiring cofactor Latrophilin 3 Lphn3 1.1911.172 0.025 GO: 0007218 neuropeptide signaling pathway GO: 0007420 braindevelopment FK506 binding protein 14 Fkbp14 1.190 1.332 0.022 GO:0006457 protein folding Nitric oxide synthase 2, Nos2 1.188 1.001 0.021GO: 0001542 ovulation from ovarian follicle; GO: 0001666 inducibleresponse to hypoxia; GO: 0001935 endothelial cell proliferation; GO:0001974 blood vessel remodeling; GO: 0006527 arginine catabolic process;GO: 0006801 superoxide metabolic process; GO: 0006809 nitric oxidebiosynthetic process; GO: 0007165 signal transduction; GO: 0007199G-protein signaling, coupled to cGMP nucleotide second messenger; GO:0007243 intracellular protein kinase cascade Olfactory receptor 1105Olr1105 1.187 −1.050 0.026 GO: 0007186 G-protein coupled receptorprotein signaling pathway; GO: 0050911 detection of chemical stimulusinvolved in sensory perception of smell Heat shock protein beta 2 Hspb21.186 −1.117 0.041 GO: 0007525 somatic muscle development GO: 0009408response to heat Uncoupling protein 1 Ucp1 1.185 1.100 0.009 GO: 0006091generation of precursor metabolites and energy; (mitochondrial, protoncarrier) GO: 0006839 mitochondrial transport; GO: 0015992 protontransport; GO: 0032870 cellular response to hormone stimulus; GO:0044253 positive regulation of multicellular organismal metabolicprocess; GO: 0048545 response to steroid hormone stimulus; GO: 0050873brown fat cell differentiation; GO: 0055085 transmembrane transportAnkyrin repeat and SOCS Asb2 1.182 1.117 0.037 box-containing 2 WDrepeat domain 31 Wdr31 1.182 1.041 0.036 Neurexophilin 4 Nxph4 1.180−1.046 0.047 Keratin 82 Krt82 1.178 1.063 0.043 Feline leukemia virusFlvcr2 1.177 −1.071 0.028 GO: 0055085 transmembrane transport subgroup Ccellular receptor family, member 2 Alpha-2u globulin PGCL4 | Obp3|Mup4|1.176 −1.185 0.030 GO: 0006810 transport major urinary protein 4 |LOC259244| alpha-2u globulin PGCL3 | LOC259246| alpha-2u globulinLOC298111| PGCL1 | alpha2u globulin | LOC298109| alpha-2u globulin PGCL2| LOC366380 alpha 2U globulin Reelin Reln 1.176 1.055 0.037 GO: 0000904cell morphogenesis involved in differentiation; GO: 0001764 neuronmigration; GO: 0007411 axon guidance; GO: 0007417 central nervous systemdevelopment; GO: 0007420 brain development; GO: 0007626 locomotorybehavior; GO: 0010001 glial cell differentiation; GO: 0018108peptidyl-tyrosine phosphorylation; GO: 0021511 spinal cord patterning;GO: 0021800 cerebral cortex tangential migration Vesicle-associatedmembrane Vamp7 1.175 1.049 0.048 GO: 0006888 ER to Golgivesicle-mediated transport; GO: protein 7 0006906 vesicle fusion; GO:0006911 phagocytosis, engulfment; GO: 0008333 endosome to lysosometransport; GO: 0015031 protein transport; GO: 0016044 cellular membraneorganization; GO: 0016192 vesicle-mediated transport; GO: 0017156calcium ion-dependent exocytosis; GO: 0043308 eosinophil degranulation;GO: 0043312 neutrophil degranulation Plasminogen Plg 1.175 −1.022 0.037GO: 0006915 apoptosis; GO: 0006917 induction of apoptosis; GO: 0007596blood coagulation; GO: 0042246 tissue regeneration; GO: 0045445 myoblastdifferentiation; GO: 0046716 muscle cell homeostasis; GO: 0048771 tissueremodeling; GO: 0051603 proteolysis involved in cellular proteincatabolic process; GO: 0051918 negative regulation of fibrinolysis; GO:0051919 positive regulation of fibrinolysis Family with sequence Fam131b1.173 1.085 0.020 similarity 131, member B Cholinergic receptor, Chrnb21.169 1.112 0.004 GO: 0001508 regulation of action potential; GO:0001661 nicotinic, beta 2 (neuronal) conditioned taste aversion; GO:0001666 response to hypoxia; GO: 0006811 ion transport; GO: 0006816calcium ion transport; GO: 0006939 smooth muscle contraction GO: 0007165signal transduction; GO: 0007271 synaptic transmission, cholinergic. GO:0007601 visual perception; GO: 0007605 sensory perception of soundDynein light chain LC8- Dynll1 1.169 1.153 0.033 GO: 0006809 nitricoxide biosynthetic process; GO: 0007017 type 1 microtubule-basedprocess; GO: 0008633 activation of pro- apoptotic gene products; GO:0042133 neurotransmitter metabolic process; GO: 0042326 negativeregulation of phosphorylation Kinesin family member 27 Kif27 1.167 1.0780.005 GO: 0007018 microtubule-based movement LOC362793 RGD1307315 1.1651.033 0.021 Unc-50 homolog Unc50 1.163 1.035 0.044 GO: 0007166 cellsurface receptor linked signaling pathway (C. elegans) GO: 0015031protein transport Sonic hedgehog Shh 1.163 1.202 0.010 GO: 0001525angiogenesis; GO: 0001569 patterning of blood vessels; GO: 0001570vasculogenesis; GO: 0001656 metanephros development; GO: 0001658branching involved in ureteric bud morphogenesis; GO: 0001666 responseto hypoxia; GO: 0001708 cell fate specification; GO: 0001755 neuralcrest cell migration; GO: 0001822 kidney development; GO: 0001841 neuraltube formation Histidine decarboxylase Hdc 1.162 1.041 0.044 GO: 0001692histamine metabolic process; GO: 0006519 cellular amino acid andderivative metabolic process; GO: 0006547 histidine metabolic process;GO: 0006548 histidine catabolic process; GO: 0019752 carboxylic acidmetabolic process; GO: 0042423 catecholamine biosynthetic processOlfactory receptor 1593 Olr1593 1.162 1.083 0.037 GO: 0007186 G-proteincoupled receptor protein signaling pathway; GO: 0050911 detection ofchemical stimulus involved in sensory perception of smell Zinc fingerprotein 354A Zfp354a 1.160 1.051 0.007 GO: 0000122 negative regulationof transcription from RNA polymerase II promoter; GO: 0001666 responseto hypoxia; GO: 0001822 kidney development; GO: 0006355 regulation oftranscription, DNA-dependent; GO: 0007275 multicellular organismaldevelopment; GO: 0007576 nucleolar fragmentation; GO: 0051593 responseto folic acid Tumor protein p63 Tp63 1.157 1.048 0.026 GO: 0000122negative regulation of transcription from RNA polymerase II promoter;GO: 0001302 replicative cell aging; GO: 0001501 skeletal systemdevelopment; GO: 0001736 establishment of planar polarity; GO: 0001738morphogenesis of a polarized epithelium; GO: 0001942 hair follicledevelopment; GO: 0002053 positive regulation of mesenchymal cellproliferation; GO: 0002064 epithelial cell development; GO: 0006915apoptosis; GO: 0006916 anti- apoptosis Histone cluster 1, Hlt Hist1hlt1.155 1.034 0.015 GO: 0006334 nucleosome assembly; GO: 0007275multicellular organismal development; GO: 0007283 spermatogenesis; GO:0007339 binding of sperm to zona pellucida; GO: 0030154 celldifferentiation; GO: 0030317 sperm motility Disabled homolog 1 Dab11.154 −1.073 0.018 GO: 0001764 neuron migration; GO: 0007162 negative(Drosophila) regulation of cell adhesion; GO: 0007264 small GTPasemediated signal transduction; GO: 0007275 multicellular organismaldevelopment; GO: 0007399 nervous system development; GO: 0007420 braindevelopment; GO: 0021589 cerebellum structural organization; GO: 0021795cerebral cortex cell migiation; GO: 0021799 cerebral cortex radiallyoriented cell migration; GO: 0021813 cell-cell adhesion involved inneuronal-glial interactions involved in cerebral cortex radial gliaguided migration Leucine rich repeat Lrrc66 1.154 1.139 0.045 containing66 Neuronal PAS domain Npas4 1.154 1.095 0.015 GO: 0007165 signaltransduction; GO: 0045941 positive protein 4 regulation oftranscription; GO: 0045944 positive regulation of transcription from RNApolymerase II promoter; GO: 0045944 positive regulation of transcriptionfrom RNA polymerase II promoter N-acetylneuraminic acid Nanp 1.154 1.1650.042 GO: 0005975 carbohydrate metabolic process; GO: 0008152phosphatase metabolic process GO: 0046380 N-acetylneuraminatebiosynthetic process MAD2L1 binding protein Mad2l1bp 1.152 1.111 0.004GO: 0007093 mitotic cell cycle checkpoint. GO: 0007096 regulation ofexit from mitosis Neuromedin S NMS 1.152 −1.015 0.045 GO: 0006940regulation of smooth muscle contraction GO: 0007218 neuropeptidesignaling pathway; GO: 0045475 locomotor rhythm Transmembrane protease,Tmprss8 1.149 1.203 0.035 GO: 0006508 proteolysis; GO: 0006811 iontransport; serine 8 (intestinal) GO: 0006814 sodium ion transportMyoglobin Mb 1.149 1.036 0.015 GO: 0001666 response to hypoxia; GO:0006810 transport; GO: 0007507 heart development; GO: 0009725 responseto hormone stimulus; GO: 0015671 oxygen transport; GO: 0031444slow-twitch skeletal muscle fiber contraction; GO: 0042542 response tohydrogen peroxide; GO: 0043353 enucleate erythrocyte differentiation;GO: 0050873 brown fat cell differentiation Similar to RIKEN cDNARGD621352 1.145 1.411 0.006 1500031L02 Dehydrogenase/reductase Dhrs71.145 −1.013 0.046 GO: 0055114 oxidation reduction (SDR family) member 7Nuclear pore associated Npap60 1.143 1.073 −0.033 GO: 0001841 neuraltube formation; GO: 0015031 protein protein transport; GO: 0046907intracellular transport; GO: 0051028 mRNA transport; GO: 0055085transmembrane transport Olfactory receptor 484 Olr484 1.141 1.355 0.016GO: 0007186 G-protein coupled receptor protein signaling pathway; GO:0050911 detection of chemical stimulus involved in sensory perception ofsmell Torsin family 3, member A Tor3a 1.140 1.088 0.020 GO: 0051085chaperone mediated protein folding requiring cofactor Similar to ProteinC20orf103 RGD1306991 1.139 −1.041 0.041 precursor Similar to RAN proteinRGD1306195 1.138 1.112 0.014 GO: 0006886 intracellular proteintransport; GO: 0006913 nucleocytoplasmic transport; GO: 0007165 signaltransduction Natriuretic peptide receptor Npr1 1.138 1.050 0.034 GO:0006182 cGMP biosynthetic process, GO: 0006468 A/guanylate cyclase Aprotein amino acid phosphorylation; GO: 0007166 cell surface(atrionatriuretic peptide receptor linked signaling pathway; GO: 0007168receptor receptor A) guanylyl cyclase signaling pathway; GO: 0008217regulation of blood pressure; GO: 0030828 positive regulation of cGMPbiosynthetic process; GO: 0042417 dopamine metabolic process Olfactoryreceptor 174 Olr174 1.138 1.294 0.040 GO: 0007186 G-protein coupledreceptor protein signaling pathway GO: 0050911 detection of chemicalstimulus involved in sensory perception of smell NADH dehydrogenaseNdufaf3 1.131 −1.084 0.042 GO: 0032981 mitochondrial respiratory chaincomplex I (ubiquinone) 1 alpha assembly I assembly subcomplex, assemblyfactor 3 Interleukin 6 signal Il6st 1.128 1.061 0.001 GO: 0005977glycogen metabolic process; GO: 0006642 transducer triglyceridemobilization; GO: 0007165 signal transduction; GO: 0007259 JAK-STATcascade; GO: 0007584 response to nutrient; GO: 0008284 positiveregulation of cell proliferation; GO: 0008593 regulation of Notchsignaling pathway; GO: 0014911 positive regulation of smooth muscle cellmigration; GO: 0019221 cytokine-mediated signaling pathway; GO: 0030307positive regulation of cell growth Synuclein, alpha (non A4 Snca 1.1271.096 0.005 GO: 0001774 microglial cell activation; GO: 0001921 positivecomponent of amyloid regulation of receptor recycling; GO: 0001956positive precursor) regulation of neurotransmitter secretion; GO:0001963 synaptic transmission, dopaminergic; GO: 0006631 fatty acidmetabolic process; GO: 0006638 neutral lipid metabolic process; GO:0006644 phospholipid metabolic process; GO: 0006916 anti- apoptosis; GO:0007006 mitochondrial membrane organization; GO: 0008344 adultlocomotory behavior Interferon regulatory factor 3 Irf3 1.127 1.1660.011 GO: 0006355 regulation of transcription, DNA-dependent; GO:0007249 I-kappaB kinase/NF-kappaB cascade; GO: 0009617 response tobacterium; GO: 0031663 lipopolysaccharide-mediated signaling pathway;GO: 0032496 response to lipopolysaccharide; GO: 0043330 response toexogenous dsRNA; GO: 0045351 type I interferon biosynthetic processAsialoglycoprotein Asgr1 1.121 1.051 0.028 GO: 0006897 endocytosis; GO:0031668 cellular response to receptor 1 extracellular stimulus Heatshock 105 kDa/ Hsph1 1.120 1.358 0.036 GO: 0006950 response to stress;GO: 0051085 chaperone 110 kDa protein 1 mediated protein foldingrequiring cofactor Actin, gamma 2, smooth Actg2 1.117 1.070 0.008 GO:0006936 muscle contraction muscle, enteric Similar to putative protein,RGD1309228 1.111 1.099 0.007 with at least 9 transmembrane domains, ofeukaryotic origin (43.9 kD) (2G415) Transient receptor potential Trpv21.111 1.060 0.018 GO: 0006811 ion transport; GO: 0006816 calcium ioncation channel, subfamily V, transport; GO: 0009266 response totemperature stimulus; member 2 GO: 0009408 response to heat; GO: 0055085transmembrane transport Glutathione 5-transferase, Gstt2 1.108 1.1230.033 GO: 0006749 glutathione metabolic process theta 2 Adenosine A3receptor Adora3 1.099 −1.150 0.048 GO: 0001973 adenosine receptorsignaling pathway; GO: 0002553 histamine secretion by mast cell; GO:0002687 positive regulation of leukocyte migration; GO: 0007165 signaltransduction; GO: 0007186 G-protein coupled receptor protein signalingpathway; GO: 0014061 regulation of norepinephrine secretion; GO: 0014068positive regulation of phosphoinositide 3-kinase cascade; GO: 0043306positive regulation of mast cell degranulation; GO: 0050729 positiveregulation of inflammatory response; GO: 0050850 positive regulation ofcalcium-mediated signaling Sarcolipin Sln 1.087 1.355 0.038 GO: 0051924regulation of calcium ion transport N-myc downstream regulated Ndrg41.087 1.105 0.010 gene 4 Gypsy retrotransposon Gin1 1.086 1.054 0.025GO: 0015074 DNA integration integrase 1 ATP-binding cassette, Abcg3l1|1.084 1.064 0.043 sub-family G (WHITE), Abcg3l2| member 3-like 1 | ATP-RGD1564709| binding cassette, sub-family LOC360997 G (WHITE), member3-like 2 | similar to ATP-binding cassette, sub-family G (WHITE), member3 Oxytocin, prepropeptide | Oxt|Avp 1.084 −1.112 0.013 GO: 0001696gastric acid secretion; GO: 0001975 response to arginine vasopressinamphetamine; GO: 0002027 regulation of heart rate; GO: 0002125 maternalaggressive behavior; GO: 0003077 negative regulation of diuresis; GO:0003079 positive regulation of natriuresis; GO: 0006950 response tostress; GO: 0007204 elevation of cytosolic calcium ion concentration;GO: 0007507 heart development; GO: 0007565 female pregnancy Thimetoligopeptidase 1 Thop1 1.080 −1.013 0.049 GO: 0006508 proteolysis; GO:0006518 peptide metabolic process GO: 0007243 intracellular proteinkinase cascade cAMP responsive element Creb3 1.078 1.137 0.021 GO:0006355 regulation of transcription, DNA-dependent binding protein 3Peroxisomal biogenesis Pex12 1.071 1.149 0.001 GO: 0007031 peroxisomeorganization. GO: 0015031 protein factor 12 transport; GO: 0016558protein import into peroxisome matrix Endothelin receptor type A |Ednra| 1.071 1.130 0.009 GO: 0001569 patterning of blood vessels; GO:0001666 endothelin-1 receptor-like LOC100366209 response to hypoxia; GO:0001701 in utero embryonic development; GO: 0001934 positive regulationof protein amino acid phosphorylation; GO: 0007165 signal transduction;GO: 0007204 elevation of cytosolic calcium ion concentration; GO:0007205 activation of protein kinase C activity by G-protein coupledreceptor protein signaling pathway; GO: 0007507 heart development; GO:0007585 respiratory gaseous exchange; GO: 0008217 regulation of bloodpressure Mk1 1.068 −1.152 0.038 GO: 0030036 actin cytoskeletonorganization Protein Pggt1b 1.062 1.162 0.039 GO: 0008284 positiveregulation of cell proliferation; GO: geranylgeranyltransferase 0018348protein amino acid geranylgeranylation; GO: type I, beta subunit 0034097response to cytokine stimulus; GO: 0045787 positive regulation of cellcycle; GO: 0051774 negative regulation of nitric-oxide synthase 2biosynthetic process; GO: 0051789 response to protein stimulus Olfactoryreceptor 1356 Olr1356 1.061 1.323 0.021 GO: 0007186 G-protein coupledreceptor protein signaling pathway; GO: 0050911 detection of chemicalstimulus involved in sensory perception of smell Immunity-related GTPaseIrgc1 1.060 −1.124 0.017 family, cinema 1 Distal-less homeobox 5 Dlx51.050 −1.140 0.008 GO: 0001649 osteoblast differentiation; GO: 0001958endochondral ossification, GO: 0006355 regulation of transcription,DNA-dependent; GO: 0007275 multicellular organismal development; GO:0007399 nervous system development; GO: 0007409 axonogenesis; GO:0007411 axon guidance; GO: 0008283 cell proliferation; GO: 0030326embryonic limb morphogenesis; GO: 0030855 epithelial celldifferentiation RELT-like 2 | FCH and Rell2|Fchsd1 1.047 −1.088 0.041GO: 0010811 positive regulation of cell-substrate adhesion double SH3domains 1 Membrane magnesium Mmgt2 1.047 1.171 0.025 GO: 0006810transport; GO: 0006824 cobalt ion transport; transporter 2 GO: 0006825copper ion transport; GO: 0006828 manganese ion transport; GO: 0015674di-, tri-valent inorganic cation transport; GO: 0015675 nickel iontransport; GO: 0015693 magnesium ion transport STEAP family member 3Steap3 1.046 1.177 0.004 GO: 0006811 ion transport; GO: 0006826 iron iontransport; GO: 0006915 apoptosis; GO: 0006917 induction of apoptosis;GO: 0007049 cell cycle; GO: 0009306 protein secretion; GO: 0055114oxidation reduction Sialidase 2 Neu2 1.030 1.134 0.023 GO: 0008152metabolic process; GO: 0010831 positive (cytosolic sialidase) regulationof myotube differentiation; GO: 0045471 response to ethanol; GO: 0045663positive regulation of myoblast differentiation Spermatogenesis Spata61.028 1.057 0.045 GO: 0007275 multicellular organismal development; GO:associated 6 0007283 spermatogenesis; GO: 0030154 cell differentiationFibroblast growth Fgf20 1.027 −1.096 0.044 GO: 0008284 positiveregulation of cell proliferation; GO: factor 20 0008543 fibroblastgrowth factor receptor signaling pathway; GO: 0016049 cell growth; GO:0030154 cell differentiation; GO: 0060113 inner ear receptor celldifferentiation Cholinergic receptor, Chnra9 1.022 −1.124 0.006 GO:0006812 cation transport; GO: 0006816 calcium ion nicotinic, alpha 9transport; GO: 0007204 elevation of cytosolic calcium ion concentration;GO: 0007605 sensory perception of sound; GO: 0042472 inner earmorphogenesis; GO: 0050910 detection of mechanical stimulus involved insensory perception of sound Coagulation factor II F2r 1.005 1.345 0.007GO: 0000186 activation of MAPKK activity; GO: 0002248 (thrombin)receptor connective tissue replacement during inflammatory response; GO:0006919 activation of caspase activity; GO: 0006954 inflammatoryresponse; GO: 0007165 signal transduction; GO: 0007186 G-protein coupledreceptor protein signaling pathway; GO: 0007205 activation of proteinkinase C activity by G-protein coupled receptor protein signalingpathway; GO: 0007260 tyrosine phosphorylation of STAT protein; GO:0007262 STAT protein nuclear translocation; GO: 0007529 establishment ofsynaptic specificity at neuromuscular junction Potassium channel, Kcnk11.004 1.241 0.015 GO: 0006811 ion transport; GO: 0006813 potassium ionsubfamily K, member 1 transport; GO: 0035094 response to nicotineAmeloblastin Ambn −1.000 −1.046 0.025 Adhesion molecule Amigo3 −1.0231.147 0.022 GO: 0007155 cell adhesion; GO: 0007157 heterophiliccell-cell with Ig like domain 3 adhesion; GO: 0007399 nervous systemdevelopment Ankyrin repeat and Asb6 −1.028 −1.072 0.015 SOCSbox-containing 6 ATPase, H transporting, Atp6v1b2 −1.039 1.046 0.011 GO:0006811 ion transport; GO: 0007035 vacuolar lysosomal V1 subunit B2acidification; GO: 0015986 ATP synthesis coupled proton transport; GO:0015992 proton transport; GO: 0030641 regulation of cellular pH; GO:0046034 ATP metabolic process Myeloid/lymphoid or Mllt10 −1.041 −1.0010.023 GO: 0008150 biological_process mixed-lineage leukemia (trithoraxhomolog, Drosophila); translocated to, 10 Phosphorylase kinase, betaPhkb −1.042 1.078 0.009 GO: 0005976 polysaccharide metabolic process;GO: 0005977 glycogen metabolic process Olfactory receptor 1409 Olr1409−1.059 −1.105 0.038 GO: 0007186 G-protein coupled receptor proteinsignaling pathway; GO: 0050911 detection of chemical stimulus involvedin sensory perception of smell MIF4G domain containing Mif4gd −1.063−1.109 0.002 GO: 0006417 regulation of translation; GO: 0016070 RNAmetabolic process Olfactory receptor 44 Olr44 −1.073 −1.122 0.039 GO:0007186 G-protein coupled receptor protein signaling pathway; GO:0050911 detection of chemical stimulus involved in sensory perception ofsmell Sulfatase modifying factor 2 Sumf2 −1.077 −1.088 0.034 WD repeatdomain 45 Wdr45 −1.078 1.120 0.049 Synclecan binding protein Sdcbp−1.080 1.170 0.008 GO: 0007265 Ras protein signal transductionBernardinelli-Seip congenital Bscls2 −1.082 −1.006 0.047 GO: 0008150biological_process lipodystrophy 2 homolog (human)Dehydrogenase/reductase Dhrs1 −1.083 1.116 0.006 GO: 0055114 oxidationreduction (SDR family) member 1 Polymerase (DNA directed), Polg −1.089−1.102 0.029 GO: 0006264 mitochondrial DNA replication GO: 0006287 gammabase-excision repair, gap-filling; GO: 0007568 aging Protein Pofut1−1.091 1.038 0.026 GO: 0001525 angiogenesis; GO: 0001756 somitogenesis;GO: O-fucosyltransferase 1 0005975 carbohydrate metabolic process; GO:0006004 fucose metabolic process; GO: 0006493 protein amino acidO-linked glycosylation; GO: 0007219 Notch signaling pathway; GO: 0007399nervous system development; GO: 0007507 heart development; GO: 0008150biological_process Outer dense fiber of sperm Odf2 −1.098 −1.005 0.044GO: 0007286 spermatid development tails 2 Nuclear prelamin A Narfl−1.099 −1.006 0.017 GO: 0001666 response to hypoxia; GO: 0016226iron-sulfur recognition factor-like cluster assembly; GO: 0032364 oxygenhomeostasis; GO: 0045449 regulation of transcription Solute carrierfamily 6 Slc6a4 −1.105 −1.065 0.038 GO: 0001504 neurotransmitter uptake;GO: 0006837 serotonin (neurotransmitter transporter, transport; GO:0007626 locomotory behavior; GO: 0009636 serotonin), member 4 responseto toxin; GO: 0015844 monoamine transport; GO: 0021794 thalamusdevelopment; GO: 0051610 serotonin uptake Acyl-Coenzyme A oxidase Acox3−1.106 −1.081 0.001 GO: 0006629 lipid metabolic process; GO: 0006631fatty acid 3, pristanoyl metabolic process; GO: 0006635 fatty acidbeta-oxidation; GO: 0033540 fatty acid beta-oxidation using acyl-CoAoxidase; GO: 0055114 oxidation reduction Shroom family member 2 Shroom2−1.115 1.191 0.035 GO: 0000902 cell morphogenesis; GO: 0007275multicellular organismal development; GO: 0016477 cell migration; GO:0030835 negative regulation of actin filament depolymerization; GO:0032438 melanosome organization. GO: 0045217 cell-cell junctionmaintenance; GO: 0051017 actin filament bundle assembly Similar to RIKENLOC498029 −1.115 −1.077 0.023 GO: 0006281 DNA repair cDNA A730011L01gene Neuropeptide FF Npffr1 −1.117 −1.111 0.024 GO: 0007165 signaltransduction GO: 0007186 G-protein receptor 1 coupled receptor proteinsignaling pathway Kelch-like 36 (Drosophila) Klhl36 −1.119 −1.021 0.014Phosphatidylinositol glycan Pigs −1.128 1.007 10.033 GO: 0006506 GPIanchor biosynthetic process; GO: 0016255 anchor biosynthesis, classattachment of GPI anchor to S protein PDZ and LIM domain 1 Pdlim1 −1.1311.111 0.049 GO: 0001666 response to hypoxia; GO: 0045449 regulation oftranscription Alpha-1-B glycoprotein Albg −1.132 −1.048 0.005 WD repeatdomain 7 Wdr7 −1.133 −1.041 0.017 Protein O-linked mannose Pomgnt1−1.135 −1.056 0.012 GO: 0006487 protein amino acid N-linkedglycosylation; beta1,2-N GO: 0006493 protein amino acid O-linkedglycosylation; acetylglucosaminyltransferase GO: 0008150biological_process Fibroblast growth factor 11 Fgf11 −1.135 −1.162 0.016DEAD (Asp-Glu-Ala-Asp) Ddx24 −1.139 1.006 0.027 box polypeptide 24Heterogeneous nuclear Hnrph1 −1.143 1.100 0.040 GO: 0006396 RNAprocessing ribonucleoprotein H1 Rho-related BTB domain Rhobtb2 −1.145−1.024 0.013 GO: 0007264 small GTPase mediated signal transductioncontaining 2 Ring finger protein 135 Rnf135 −1.147 −1.045 0.013 GO:0006270 DNA-dependent DNA replication initiation; GO: 0007264 smallGTPase mediated signal transduction; GO: 0047497 mitochondrion transportalong microtubule Vaccinia related kinase 3 Vrk3 −1.150 1.011 0.046 GO:0006468 protein amino acid phosphorylation; GO: 0032516 positiveregulation of phosphoprotein phosphatase activity; GO: 0070373 negativeregulation of ERK1 and ERK2 cascade Cardiotrophin-like cytokine Clcf1−1.150 −1.144 0.029 GO: 0007166 cell surface receptor linked signalingpathway; factor 1 GO: 0007259 JAK-STAT cascade; GO: 0030183 B celldifferentiation Prostate tumor overexpressed Ptov1 −1.154 −1.014 0.050GO: 0045449 regulation of transcription 1 Mitogen activated proteinMap3k1 −1.158 −1.106 0.008 GO: 0000165 MAPKKK cascade; GO: 0000186activation of kinase kinase kinase 1 MAPKK activity; GO: 0000209 proteinpolyubiquitination; GO: 0006468 protein amino acid phosphorylation; GO:0006970 response to osmotic stress; GO: 0007179 transforming growthfactor beta receptor signaling pathway; GO: 0007249 I-kappaBkinase/NF-kappaB cascade; GO: 0007254 JNK cascade; GO: 0007256activation of JNKK activity; GO: 0007257 activation of JUN kinaseactivity Calcium homeostasis Cherp| −1.160 −1.040 0.018 GO: 0006396 RNAprocessing; GO: 0006874 cellular calcium endoplasmic reticulumRGD1311847 ion homeostasis; GO: 0008285 negative regulation of cellprotein | similar to proliferation 1700030K09Rik protein Progestin andadipoQ receptor Paqr3 −1.161 −1.063 0.016 family member III Glioma tumorsuppressor Gltscr2 −1.162 −1.123 0.012 candidate region gene 2 Poly(rC)binding protein 2 Pcbp2 −1.162 −1.015 0.014 GO: 0008380 RNA splicing THOcomplex 5 Thoc5 −1.163 −1.025 0.036 GO: 0006397 mRNA processing; GO:0006406 mRNA export from nucleus; GO: 0006810 transport; GO: 0008150biological_process; GO: 0008380 RNA splicing; GO: 0030154 celldifferentiation; GO: 0045650 negative regulation of macrophagedifferentiation; GO: 0046784 intronless viral mRNA export from hostnucleus; GO: 0051028 mRNA transport Glycine-, glutamate-, LOC246295−1.165 1.035 0.045 thienylcyclohexylpiperidine- binding protein Familywith sequence Fam113a −1.171 1.022 0.049 similarity 113, member ALeucine rich repeat (in FLII) Lrrfip1 −1.175 1.000 0.048 GO: 0045449regulation of transcription interacting protein 1 AlkB, alkylationrepair Alkbh3 −1.177 −1.138 0.001 GO: 0006281 DNA repair; GO: 0055114oxidation reduction homolog 3 (E. coli) Amyloid beta (A4) precursorApbb3 −1.181 1.016 0.018 protein-binding, family B, member 3Cyclin-dependent kinase Cdkn2b −1.185 −1.104 0.017 GO: 0000079regulation of cyclin-dependent protein kinase inhibitor 2B (p15,inhibits activity; GO: 0000086 G2/M transition of mitotic cell cycle;CDK4) GO: 0007050 cell cycle arrest; GO: 0008285 negative regulation ofcell proliferation; GO: 0014070 response to organic cyclic substance;GO: 0030219 megakalyocyte differentiation; GO: 0030511 positiveregulation of transforming growth factor beta receptor signalingpathway; GO: 0030858 positive regulation of epithelial celldifferentiation; GO: 0031575 G1/S transition checkpoint; GO: 0031668cellular response to extracellular stimulus CD2-associated protein Cd2ap−1.189 −1.089 0.039 GO: 0016337 cell-cell adhesion; GO: 0016477 cellmigration; GO: 0043161 proteasomal ubiquitin-dependent protein catabolicprocess; GO: 0048259 // regulation of receptor- mediated endocytosisATPase, Na+/K+ transporting, Atp1b1 −1.192 −1.020 0.036 GO: 0001666response to hypoxia; GO: 0006754 ATP beta 1 polypeptide biosyntheticprocess; GO: 0006811 ion transport; GO: 0006813 potassium ion transport;GO: 0006814 sodium ion transport; GO: 0030001 metal ion transport Ringfinger protein 114 Rnf114 −1.193 1.005 0.050 GO: 0007275 multicellularorganismal development; GO: 0007283 spermatogenesis; GO: 0030154 celldifferentiation Inositol polyphosphate Inppl1 −1.198 −1.022 0.046 GO:0006006 glucose metabolic process; GO: 0007420 brain phosphatase-like 1development; GO: 0008156 negative regulation of DNA replication; GO:0008285 negative regulation of cell proliferation; GO: 0009791post-embryonic development; GO: 0010629 negative regulation of geneexpression; GO: 0010642 negative regulation of platelet-derived growthfactor receptor signaling pathway; GO: 0010977 negative regulation ofneuron projection development; GO: 0032868 response to insulin stimulus;GO: 0032957 inositol trisphosphate metabolic process TAO kinase 2 Taok2−1.200 −1.058 0.010 GO: 0000186 activation of MAPKK activity; GO:0006468 protein amino acid phosphmylation; GO: 0006950 response tostress; GO: 0008360 regulation of cell shape; GO: 0030036 actincytoskeleton organization; GO: 0046330 positive regulation of INKcascade; GO: 0048041 focal adhesion assembly; GO: 0000186 activation ofMAPKK activity Dynamin 2 Dnm2 −1.200 −1.085 0.036 GO: 0006892 post-Golgivesicle-mediated transport; GO: 0006897 endocytosis; GO: 0006898receptor-mediated endocytosis; GO: 0016044 cellular membraneorganization; GO: 0031623 receptor internalization; GO: 0033572transferrin transport Calcium channel, voltage- Cacnb3 −1.200 1.0090.029 GO: 0006811 ion transport; GO: 0006816 calcium ion dependent, beta3 subunit transport; GO: 0050852 T cell receptor signaling pathwaySimilar to chromosome 1 RGD1303271 −1.201 −1.049 0.048 open readingframe 172 Pre-B-cell leukemia Pbx2 −1.202 −1.007 0.019 GO: 0006355regulation of transcription, DNA-dependent, homeobox 2 GO: 0009954proximal/distal pattern formation; GO: 0030326 embryonic limbmorphogenesis; GO: 0045944 positive regulation of transcription from RNApolymerase II promoter SCY1-like 1 Scyl1|Ltbp3 −1.204 1.032 0.034 GO:0006468 protein amino acid phosphorylation; GO: (S. cerevisiae) |0006890 retrograde vesicle-mediated transport, Golgi to ER; latenttransforming GO: 0016192 vesicle-mediated transport growth factor betabinding protein 3 Olfactory receptor 1765 Olr1765 −1.206 −1.436 0.003GO: 0007186 G-protein coupled receptor protein signaling pathway; GO:0050911 detection of chemical stimulus involved in sensory perception ofsmell Transmembrane BAX Tmbim6 −1.209 −1.048 0.006 GO: 0007283spermatogenesis; GO: 0030324 lung inhibitor motif containingdevelopment; GO: 0043066 negative regulation of apoptosis 6 Solutecarrier organic anion Slco2b1 −1.210 1.027 0.030 GO: 0001889 liverdevelopment; GO: 0006811 ion transport; transporter family, member GO:0015721 bile acid and bile salt transport; GO: 0071718 2b1sodium-independent icosanoid transport Trace amine-associated Taar8c−1.210 1.130 0.011 GO: 0007165 signal transduction; GO: 0007186G-protein receptor 8c coupled receptor protein signaling pathwayTransmembrane protein, Tpra1 −1.211 −1.019 0.033 adipocyte asscociated 1DiGeorge syndrome critical Dgcr2 −1.211 −1.046 0.036 GO: 0042493response to drug region gene 2 Casein kinase 1, delta | solute Csnk1d|−1.211 −1.042 0.021 GO: 0000278 mitotic cell cycle; GO: 0006468 proteinamino carrier family 16, Slc16a3 acid phosphorylation; GO: 0032436positive regulation of member 3 (monocarboxylic proteasomalubiquitin-dependent protein catabolic process; acid transporter 4) GO:0032922 circadian regulation of gene expression; GO: 0042752 regulationof circadian rhythm; GO: 0006090 pyruvate metabolic process; GO: 0015711organic anion transport; GO: 0055085 transmembrane transport Scaffoldattachment factor B Safb −1.219 −1.003 0.016 GO: 0006355 regulation oftranscription, DNA-dependent; GO: 0030520 estrogen receptor signalingpathway; GO: 0040007 growth; GO: 0042445 hormone metabolic process; GO:0050684 regulation of mRNA processing MAP/microtubule affinity- Mark2−1.219 −1.025 0.044 GO: 0006468 protein amino acid phosphorylation; GO:regulating kinase 2 0006979 response to oxidative stress; GO: 0007243intracellular protein kinase cascade; GO: 0007275 multicellularorganismal development; GO: 0030154 cell differentiation; GO: 0045197establishment or maintenance of epithelial cell apical/basal polarityEra (G-protein)-like 1 Eral1 −1.219 1.006 0.040 (E. coli) RT1 class I,locus CE12 | RT1- −1.220 −1.156 0.002 GO: 0002474 antigen processing andpresentation of peptide RT1 class I, locus1 | RT1 CE12|RT1- antigen viaMHC class I; GO: 0006955 immune response; class I, locus CE14 CE14 GO:0019882 antigen processing and presentation Serine incorporator 3Serinc3 −1.220 1.055 0.021 GO: 0006658 phosphatidylserine metabolicprocess; GO: 0006665 sphingolipid metabolic process; GO: 0006917induction of apoptosis; GO: 0015825 L-serine transport; GO: 0051347positive regulation of transferase activity Cancer susceptibility Casc3−1.225 −1.024 0.048 GO: 0000184 nuclear-transcribed mRNA catabolicprocess, candidate 3 nonsense-mediated decay; GO: 0006397 mRNAprocessing; GO: 0006417 regulation of translation; GO: 0006810transport; GO: 0006950 response to stress; GO: 0008298 intracellularmRNA localization; GO: 0008380 RNA splicing; GO: 0051028 mRNA transportDAB2 interacting protein Dab2ip −1.233 −1.016 0.038 GO: 0007165 signaltransduction; GO: 0051056 regulation of small GTPase mediated signaltransduction Gamma-glutamyl Ggt6 −1.233 −1.024 0.018 GO: 0006750glutathione biosynthetic process transferase 6 Fatty acid desaturase 1Fads1 −1.233 −1.192 0.021 GO: 0006636 unsaturated fatty acidbiosynthetic process; GO: 0006810 transport; GO: 0006950 response tostress; GO: 0007568 aging; GO: 0007584 response to nutrient; GO: 0009267cellular response to starvation; GO: 0009744 response to sucrosestimulus; GO: 0010033 response to organic substance; GO: 0014070response to organic cyclic substance; GO: 0019369 arachidonic acidmetabolic process PTK2B protein tyrosine Ptk2b −1.233 −1.101 0.009 GO:0000165 MAPKKK cascade; GO: 0000302 response to kinase 2 beta reactiveoxygen species; GO: 0001525 angiogenesis; GO: 0001556 oocyte maturation;GO: 0001666 response to hypoxia; GO: 0006468 protein amino acidphosphorylation; GO: 0006800 oxygen and reactive oxygen speciesmetabolic process; GO: 0006950 response to stress; GO: 0006970 responseto osmotic stress; GO: 0007015 actin filament organization RAD23 homologA Rad23a −1.234 −1.089 0.008 GO: 0006289 nucleotide-excision repair; GO:0006974 (S. cerevisiae) response to DNA damage stimulus; GO: 0043161proteasomal ubiquitin-dependent protein catabolic process GDPdissociation Gdi1 −1.235 −1.013 0.025 GO: 0007264 small GTPase mediatedsignal transduction; inhibitor 1 GO: 0015031 protein transport; GO:0043087 regulation of GTPase activity Solute carrier family Slc15a4−1.237 −1.100 0.029 GO: 0006857 oligopeptide transport; GO: 0015031protein 15, member 4 transport; GO: 0015817 histidine transport DALRanticodon binding Dalrd3 −1.242 1.008 0.026 GO: 0006420 arginyl-tRNAaminoacylation domain containing 3 Axin 1 Axin1 −1.244 −1.077 0.006 GO:0007275 multicellular organismal development; GO: 0007605 sensoryperception of sound; GO: 0010800 positive regulation ofpeptidyl-threonine phosphorylation; GO: 0016055 Wnt receptor signalingpathway; GO: 0030163 protein catabolic process; GO: 0030178 negativeregulation of Wnt receptor signaling pathway; GO: 0031398 positiveregulation of protein ubiquitination; GO: 0033138 positive regulation ofpeptidyl-serine phosphorylation; GO: 0046330 positive regulation of JNKcascade; GO: 0060070 Wnt receptor signaling pathway through beta-cateninATG7 autophagy related 7 Atg7 −1.247 −1.107 0.010 GO: 0001889 liverdevelopment; GO: 0006497 protein amino homolog (S. cerevisiae) acidlipidation; GO: 0006520 cellular amino acid metabolic process; GO:0006914 autophagy; GO: 0006996 organelle organization; GO: 0007628 adultwalking behavior; GO: 0008152 metabolic process; GO: 0009791post-embryonic development; GO: 0015031 protein transport; GO: 0016044cellular membrane organization General transcription Gtf2i −1.247 1.0330.046 GO: 0009790 embryonic development GO: 0051481 reduction factor III of cytosolic calcium ion concentration Phosphatidylinositol glycanPigv −1.253 1.010 0.005 GO: 0006506 GPI anchor biosynthetic process; GO:0016254 anchor biosynthesis, class preassembly of GPI anchor in ER Vmembrane Anterior phairuc defective Aph1a −1.255 −1.029 0.046 GO:0001656 metanephros development; GO: 0006509 1 homolog A (C. elegans)membrane protein ectodomain proteolysis; GO: 0006915 apoptosis; GO:0007220 Notch receptor processing; GO: 0008624 induction of apoptosis byextracellular signals; GO: 0016485 protein processing; GO: 0031293membrane protein intracellular domain proteolysis; GO: 0042987 amyloidprecursor protein catabolic process; GO: 0043085 positive regulation ofcatalytic activity Purinergic receptor P2X, P2rx4 −1.260 −1.135 0.017GO: 0002028 regulation of sodium ion transport; GO: 0006809 ligand-gatedion channel 4 nitric oxide biosynthetic process; GO: 0006810 tiansport;GO: 0006811 ion transport; GO: 0006816 calcium ion transport; GO:0007165 signal transduction; GO: 0008217 regulation of blood pressure;GO: 0010524 positive regulation of calcium ion transport into cytosol;GO: 0010614 negative regulation of cardiac muscle hypertrophy; GO:0019228 regulation of action potential in neuron Secretory carriermembrane Scamp2 −1.262 1.071 0.018 GO: 0006886 intracellular proteintransport; GO: 0006897 protein 2 endocytosis. GO: 0015031 proteintransport Bcl2-like 1 Bcl2l1 −1.264 −1.205 0.024 GO: 0001541 ovarianfollicle development; GO: 0001666 response to hypoxia; GO: 0001701 inutero embryonic development; GO: 0001836 release of cytochrome c frommitochondria; GO: 0006915 apoptosis; GO: 0006916 anti- apoptosis; GO:0006950 response to stress; GO: 0006979 response to oxidative stress;GO: 0007281 germ cell development; GO: 0007283 spermatogenesis Olfactoryreceptor 1614 Olr1614 −1.267 −1.411 0.004 GO: 0007186 G-protein coupledreceptor protein signaling pathway; GO: 0050911 detection of chemicalstimulus involved in sensory perception of smell LUC7-like (S.cerevisiae) Luc7l −1.270 1.061 0.043 Telomerase associated Tep1 −1.271−1.012 0.016 GO: 0000722 telomere maintenance via recombination; GO:protein 1 0000722 telomere maintenance via recombination; GO: 0007004telomere maintenance via telomerase Serine/threonine kinase 39, Stk39−1.272 −1.061 0.030 GO: 0006468 protein amino acid phosphorylation; GO:STE20/SPS1 homolog (yeast) 0043268 positive regulation of potassium iontransport CREB binding protein Crebbp −1.272 −1.102 0.002 GO: 0006355regulation of transcription, DNA-dependent; GO: 0008283 cellproliferation; GO: 0016573 histone acetylation; GO: 0018076 N-terminalpeptidyl-lysine acetylation; GO: 0030718 germ-line stem cellmaintenance; GO: 0033261 regulation of S phase; GO: 0045449 regulationof transcription; GO: 0045893 positive regulation of transcription,DNA-dependent; GO: 0045941 positive regulation of transcription; GO:0045944 positive regulation of transcription from RNA polymerase IIpromoter Serine/threonine kinase 40 Stk40 −1.272 −1.073 0.016 GO:0006468 protein amino acid phosphorylation Protein kinase N1 Pkn1 −1.274−1.095 0.035 GO: 0006468 protein amino acid phosphorylation; GO: 0006972hyperosmotic response; GO: 0007165 signal transduction Ligase III, DNA,Lig3 −1.276 −1.089 0.035 GO: 0006260 DNA replication; GO: 0006281 DNArepair; ATP-dependent GO: 0006310 DNA recombination; GO: 0033151 V(D)Jrecombination Post-GPI attachment to Pgap2 −1.277 1.012 0.026 GO:0006916 anti-apoptosis; GO: 0006974 response to DNA proteins 2 damagestimulus GO: 0042770 DNA damage response, signal transduction GlucagonGcg −1.280 −1.140 0.021 GO: 0006109 regulation of carbohydrate metabolicprocess; GO: 0007186 G-protein coupled receptor protein signalingpathway; GO: 0007188 G-protein signaling, coupled to cAMP nucleotidesecond messenger; GO: 0009755 hormone-mediated signaling pathway; GO:0019216 regulation of lipid metabolic process; GO: 0019538 proteinmetabolic process; GO: 0032099 negative regulation of appetite; GO:0050796 regulation of insulin secretion CDC42 small effector 1 Cdc42se1−1.280 −1.105 0.010 GO: 0006909 phagocytosis; GO: 0008360 regulation ofcell shape Solute carrier family 25 Slc25a3 −1.282 1.010 0.025 GO:0055085 transmembrane transport (mitochondrial carrier, phosphatecarrier), member 3 Ubiquitin-associated Ubap1 −1.284 −1.090 0.006protein 1 ATP citrate lyase Acly −1.292 −1.138 0.025 GO: 0006084acetyl-CoA metabolic processt assay; GO: 0006085 acetyl-CoA biosyntheticprocess; GO: 0006101 citrate metabolic process; GO: 0006629 lipidmetabolic process; GO: 0006633 fatty acid biosynthetic process; GO:0008152 metabolic process; GO: 0044262 cellular carbohydrate metabolicprocess Potassium channel, subfamily Kcnk6| −1.294 1.142 0.019 GO:0006811 ion transport; GO: 0006813 potassium ion K, member 6 | CWC15Cwc15| transport; GO: 0000398 nuclear mRNA splicing, viaspliceosome-associated Fam35a spliceosome; GO: 0008150biological_process; GO: 0008380 protein homolog RNA splicing (S.cerevisiae) | family with sequence similarity 35, member A Inositol(myo)-1(or 4)- Impa2 −1.294 −1.024 0.018 GO: 0008150 biological_process;GO: 0046855 inositol monophosphatase 2 phosphate dephosphorylationTransmembrane protein 171 Tmem171 −1.296 1.129 0.022 Zinc finger,DHHC-type Zdhhc23 −1.301 −1.025 0.002 containing 23 A kinase (PRKA)anchor Akap1 −1.301 1.061 0.022 GO: 0010614 negative regulation ofcardiac muscle protein 1 hypertrophy; GO: 0010738 regulation of proteinkinase A signaling cascade; GO: 0032869 cellular response to insulinstimulus; GO: 0035308 negative regulation of protein amino aciddephosphorylation; GO: 0051534 negative regulation of NFAT proteinimport into nucleus; GO: 0070887 cellular response to chemical stimulusShisa homolog 5 Shisa5 −1.302 1.022 0.010 GO: 0006915 apoptosis GO:0006917 induction of apoptosis; (Xenopus laevis) GO: 0043123 positiveregulation of I-kappaB kinase Phosphatidic acid Ppap2c −1.306 −1.1450.048 phosphatase type 2c Nuclear RNA export Nxfl −1.307 1.174 0.026 GO:0006405 RNA export from nucleus; GO: 0006406 mRNA factor 1 export fromnucleus; GO: 0006810 transport; GO: 0016973 poly(A)+ mRNA export fromnucleus; GO: 0051028 mRNA transport Fas-activated serine/ Fastk −1.308−1.082 0.001 GO: 0006915 apoptosis threonine kinase Ring finger protein160 Rnf160 −1.317 1.091 0.042 PCTAIRE protein kinase 1 Pctk1 −1.318−1.126 0.029 GO: 0006468 protein amino acid phosphorylation Pricklehomolog 3 Prickle3 −1.326 −1.096 0.042 (Drosophila) Solute carrierfamily 9 Slc9a3 −1.333 1.233 0.025 GO: 0002028 regulation of sodium iontransport; GO: 0006812 (sodium/hydrogen exchanger), cation transport;GO: 0006814 sodium ion transport; GO: member 3 0006885 regulation of pH;GO: 0006898 receptor-mediated endocytosis; GO: 0007623 circadian rhythm;GO: 0051384 response to glucocorticoid stimulus; GO: 0055085transmembrane transport CDP-diacylglycerol Cds1 −1.339 −1.064 0.014 GO:0008654 phospholipid biosynthetic process synthase 1 Trinucleotiderepeat Tnrc6b −1.342 −1.089 0.040 containing 6B Cytochrome P450, family2, Cyp2d4| −1.347 −1.127 0.001 GO: 0008202 steroid metabolic process;GO: 0009804 subfamily d, Cyp2d5 coumarin metabolic process; GO: 0009820alkaloid polypeptide 4 | cytochrome metabolic process; GO: 0009822alkaloid catabolic P450, family 2, subfamily d, process; GO: 0009892negative regulation of metabolic polypeptide 5 process; GO: 0010033response to organic substance; GO: 0016098 monoterpenoid metabolicprocess; GO: 0017144 drug metabolic process; GO: 0019369 arachidonicacid metabolic process; GO: 0033076 isoquinoline alkaloid metabolicprocess Hypothetical protein RGD735175 −1.347 −1.059 0.021 MGC: 72616Basic helix-loop-helix Bhlhe40 −1.347 −1.158 0.022 GO: 0007399 nervoussystem development; GO: 0007623 family, member e40 circadian rhythm; GO:0009416 response to light stimulus; GO: 0009649 entrainment of circadianclock; GO: 0045892 negative regulation of transcription, DNA-dependent;GO: 0048168 regulation of neuronal synaptic plasticityPhosphatidylinositol 4-kinase, Pi4ka −1.348 −1.089 0.009 GO: 0046854phosphoinositide phosphorylation; GO: 0048015 catalytic, alphaphosphoinositide-mediated signaling Similar to 2310044H10Rik MGC93975−1.351 −1.168 0.001 protein CDP-diacylglycerol synthase Cds2 −1.3541.015 0.031 GO: 0008654 phospholipid biosynthetic process (phosphatidatecytidylyltransferase) 2 Prolyl endopeptidase-like Prepl −1.354 1.0060.045 GO: 0006508 proteolysis B-cell translocation gene 1, Btgl −1.360−1.130 0.043 GO: 0006479 protein amino acid methylation; GO: 0006979anti-proliferative response to oxidative stress; GO: 0007283spermatogenesis; GO: 0007286 spermatid development; GO: 0008285 negativeregulation of cell proliferation; GO: 0042981 regulation of apoptosis;GO: 0043434 response to peptide hormone stimulus; GO: 0045603 positiveregulation of endothelial cell differentiation; GO: 0045663 positiveregulation of myoblast differentiation; GO: 0045766 positive regulationof angiogenesis Claudin 3 Cldn3 −1.373 −1.030 0.031 GO: 0001666 responseto hypoxia; GO: 0016338 calcium- independent cell-cell adhesionSerine/threonine kinase 38 Stk38 −1.375 −1.058 0.006 GO: 0006464 proteinmodification process; GO: 0006468 protein amino acid phosphorylation;GO: 0007243 intracellular protein kinase cascade; GO: 0008150biological_process Mucin and cadherin like Mucdhl −1.375 −1.064 0.009GO: 0007155 cell adhesion Thyroid hormone receptor Trip10 −1.377 −1.1030.011 GO: 0006897 endocytosis; GO: 0042538 hyperosmotic salinityinteractor 10 response Solute carrier family 44, Slc44a1 −1.381 −1.0430.010 GO: 0015871 choline transport member 1 Mitofusin 2 Mfn2 −1.382−1.103 0.034 GO: 0001825 blastocyst formation GO: 0006626 proteintargeting to mitochondrion; GO: 0007006 mitochondrial membraneorganization; GO: 0007050 cell cycle arrest; GO: 0008053 mitochondrialfusion; GO: 0008285 negative regulation of cell proliferation; GO:0046580 negative regulation of Ras protein signal transduction; GO:0048593 camera-type eye morphogenesis; GO: 0048662 negative regulationof smooth muscle cell proliferation; GO: 0051646 mitochondrionlocalization Solute carrier family 9 Slc9a3r1 −1.388 −1.041 0.037 GO:0016055 Wnt receptor signaling pathway GO: 0030643 (sodium/hydrogenexchanger), cellular phosphate ion homeostasis member 3 regulator 1ATP-binding cassette, sub- Abcb6 −1.390 1.002 0.008 GO: 0006810transport; GO: 0055085 transmembrane family B (MDR/TAP), transportmember 6 Spermatogenesis associated 2 Spata2 −1.390 −1.165 0.039Sphingomyelin synthase 2 Sgms2 −1.398 −1.072 0.022 GO: 0006629 lipidmetabolic process; GO: 0006665 sphingolipid metabolic process; GO:0006686 sphingomyelin biosynthetic process Ras homolog gene family,Rhot2 −1.404 1.018 0.049 GO: 0006915 apoptosis; GO: 0007264 small GTPasemediated member T2 signal transduction; GO: 0019725 cellularhomeostasis; GO: 0047497 mitochondrion transport along microtubuleV-erb-b2 erythroblastic Erbb2 −1.417 −1.065 0.047 GO: 0001889 liverdevelopment; GO: 0007165 signal leukemia viral oncogene transduction;GO: 0007166 cell surface receptor linked homolog 2, neuro/glioblastomasignaling pathway; GO: 0007169 transmembrane receptor derived oncogenehomolog protein tyrosine kinase signaling pathway; GO: (avian) 0007399nervous system development; GO: 0007417 central nervous systemdevelopment; GO: 0007422 peripheral nervous system development; GO:0007507 heart development; GO: 0007519 skeletal muscle tissuedevelopment; GO: 0007528 neuromuscular junction development Integrinalpha FG-GAP Itfg3 −1.420 −1.003 0.039 repeat containing 3 Endothelin 2Edn2 −1.424 −1.091 0.001 GO: 0001516 prostaglandin biosynthetic process;GO: 0001543 ovarian follicle rupture; GO: 0002690 positive regulation ofleukocyte chemotaxis; GO: 0003100 regulation of systemic arterial bloodpressure by endothelin; GO: 0007204 elevation of cytosolic calcium ionconcentration; GO: 0007205 activation of protein kinase C activity by G-protein coupled receptor protein signaling pathway; GO: 0008217regulation of blood pressure; GO: 0008284 positive regulation of cellproliferation; GO: 0010460 positive regulation of heart rate; GO:0014824 artery smooth muscle Ring finger protein 10 Rnf10 −1.440 −1.0690.013 GO: 0008150 biological_process; GO: 0045941 positive regulation oftranscription Aldolase A, fructose- Aldoa −1.442 1.024 0.016 GO: 0001666response to hypoxia; GO: 0006000 fructose bisphosphate metabolicprocess; GO: 0006096 glycolysis; GO: 0006754 ATP biosynthetic process;GO: 0006941 striated muscle contraction; GO: 0008152 metabolic process;GO: 0008360 regulation of cell shape; GO: 0009408 response to heat; GO:0030388 fructose 1,6-bisphosphate metabolic process; GO: 0032496response to lipopolysaccharide Adenylate cyclase 6 Adcy6 −1.442 −1.0310.036 GO: 0006171 cAMP biosynthetic process; GO: 0007193 inhibition ofadenylate cyclase activity by G-protein signaling pathway; GO: 0009755hormone-mediated signaling pathway; GO: 0034199 activation of proteinkinase A activity UDP-glucose ceramide Ugcg −1.443 −1.257 0.001 GO:0006665 sphingolipid metabolic process GO: 0008610 glucosyltransferaselipid biosynthetic process Nuclear receptor subfamily Nr3c2 −1.447−1.092 0.009 GO: 0006883 cellular sodium ion homeostasis; GO: 0007588 3,group C, member 2 excretion, GO: 0031959 mineralocorticoid receptorsignaling pathway; GO: 0042127 regulation of cell proliferation Inositolpolyphosphate-5- Inpp5j −1.455 −1.112 0.023 GO: 0010977 negativeregulation of neuron projection phosphatase J development; GO: 0031115negative regulation of microtubule polymerization; GO: 0033137 negativeregulation of peptidyl- serine phosphorylation cytokine-like nuclearfactor N-pac −1.456 −1.106 0.032 GO: 0006098 pentose-phosphate shunt;GO: 0055114 oxidation n-pac reduction Glutamic-oxaloacetic Got2 −1.458−1.135 0.017 GO: 0006103 2-oxoglutarate metabolic process; GO: 0006107transaminase 2, mitochondrial oxaloacetate metabolic process; GO:0006520 cellular amino (aspartate aminotransferase 2) acid metabolicprocess; GO: 0006531 aspartate metabolic process; GO: 0006532 aspartatebiosynthetic process; GO: 0006533 aspartate catabolic process; GO:0006536 glutamate metabolic process; GO: 0009058 biosynthetic process;GO: 0015908 fatty acid transport; GO: 0019550 glutamate catabolicprocess to aspartate MAX interactor 1 Mxi1 −1.466 −1.047 0.018 GO:0000122 negative regulation of transcription from RNA polymerase IIpromoter; GO: 0006355 regulation of transcription, DNA-dependentVacuolar protein sorting 52 Vps52 −1.478 −1.160 0.025 GO: 0015031protein transport homolog (S. cerevisiae) Adenosylhomocysteinase Ahcy−1.487 −1.135 0.008 GO: 0001666 response to hypoxia; GO: 0002439 chronicinflammatory response to antigenic stimulus; GO: 0006730 one-carbonmetabolic process; GO: 0007584 response to nutrient; GO: 0008152metabolic process; GO: 0019510 S-adenosylhomocysteine catabolic process;GO: 0042745 circadian sleep/wake cycle Meprin 1 alpha Mep1a −1.502−1.200 0.037 GO: 0006508 proteolysis Myosin IE Myole −1.516 −1.229 0.004GO: 0001570 vasculogenesis; GO: 0001701 in utero embryonic development;GO: 0001822 kidney development; GO: 0006807 nitrogen compound metabolicprocess; GO: 0030097 hemopoiesis; GO: 0035166 post-embryonichemopoiesis; GO: 0048008 platelet-derived growth factor receptorsignaling pathway Lymphocyte antigen 6 Ly6e −1.541 −1.316 0.001 GO:0001701 in utero embryonic development; GO: 0030325 complex, locus Eadrenal gland development; GO: 0035265 organ growth; GO: 0042415norepinephrine metabolic process; GO: 0048242 epinephrine secretion; GO:0055010 ventricular cardiac muscle tissue morphogenesis Aldehydedehydrogenase 1 Aldh1a1 −1.553 1.170 0.023 GO: 0001822 kidneydevelopment; GO: 0001889 liver family, member A1 development; GO:0002072 optic cup morphogenesis involved in camera-type eye development;GO: 0006979 response to oxidative stress; GO: 0007494 midgutdevelopment; GO: 0014070 response to organic cyclic substance; GO:0032355 response to estradiol stimulus; GO: 0032526 response to retinoicacid; GO: 0042493 response to drug; GO: 0042572 retinol metabolicprocess Olfactory, receptor 434 Olr434 −1.560 −1.382 0.000 GO: 0007186G-protein coupled receptor protein signaling pathway Olfactory, receptor1607 Olr1607 −1.578 −1.370 0.043 GO: 0007186 G-protein coupled receptorprotein signaling pathway; GO: 0050911 detection of chemical stimulusinvolved in sensory perception of smell Serine peptidase inhibitor,Spint1 −1.611 −1.093 0.011 GO: 0001763 morphogenesis of a branchingstructure; GO: Kunitz type 1 0001892 embryonic placenta development; GO:0030198 extracellular matrix organization; GO: 0060670 branchinginvolved in embryonic placenta morphogenesis; GO: 0060674 placenta bloodvessel development D site of albumin promoter Dbp −1.647 −1.918 0.000GO: 0006355 regulation of transcription, DNA-dependent (albumin D-box)binding GO: 0042127 regulation of cell proliferation; GO: 0048511protein rhythmic process Solute carrier family 5 Slc5a6 −1.650 −1.2220.021 GO: 0006811 ion transport GO: 0006814 sodium ion transport;(sodium-dependent vitamin GO: 0015878 biotin transport; GO: 0015887pantothenate transporter), member 6 transmembrane transport; GO: 0055085transmembrane transport Multiple inositol Minpp1 −1.658 −1.225 0.010 GO:0048015 phosphoinositide-mediated signaling polyphosphate histidinephosphatase 1 Tsukushin Tsku −1.682 1.012 0.014 Solute carrier family26, Slc26a3 −1.724 −1.112 0.034 GO: 0006820 anion transport; GO: 0008272sulfate transport; member 3 GO: 0055085 transmembrane transportCystathionase (cystathionine Cth −1.725 −1.172 0.046 GO: 0006520cellular amino acid metabolic process; GO: gamma-lyase) 0006749glutathione metabolic process; GO: 0008285 negative regulation of cellproliferation; GO: 0008652 cellular amino acid biosynthetic process; GO:0018272 protein-pyridoxal-5- phosphate linkage via peptidyl-N6-pyridoxalphosphate-L- lysine; GO: 0019344 cysteine biosynthetic process; GO:0019346 transsulfuration // traceable author statement; GO: 0030308negative regulation of cell growth; GO: 0050667 homocysteine metabolicprocess; GO: 0051289 protein homotetramerization Peripheral myelinprotein Pmp22 −1.955 −1.443 0.042 GO: 0007049 cell cycle; GO: 0007050cell cycle arrest; GO: 22 0008285 negative regulation of cellproliferation; GO: 0030154 cell differentiation; GO: 0032288 myelinassembly; GO: 0042552 myelination Hydroxysteroid 11-beta Hsd11b2 −2.0021.001 0.041 GO: 0001666 response to hypoxia; GO: 0002017 regulation ofdehydrogenase 2 blood volume by renal aldosterone; GO: 0006950 responseto stress; GO: 0007565 female pregnancy; GO: 0008152 metabolic process;GO: 0008211 glucocorticoid metabolic process; GO: 0032094 response tofood; GO: 0032868 response to insulin stimulus; GO: 0042493 response todrug; GO: 0048545 response to steroid hormone stimulus Hydroxysteroid(17-beta) Hsd17b2 −2.530 −1.603 0.040 GO: 0006694 steroid biosyntheticprocess; GO: 0032526 dehydrogenase 2 response to retinoic acid; GO:0055114 Nuclear receptor subfamily 1, Nr1d1|Thra −2.844 −2.072 0.018 GO:0006355 regulation of transcription, DNA-dependent; group D, member 1 |thyroid GO: 0007623 circadian rhythm; GO: 0001502 cartilage hormonereceptor alpha condensation; GO: 0001503 ossification; GO: 0001822kidney development; GO: 0001889 liver development; GO: 0002155regulation of thyroid hormone mediated signaling pathway; GO: 0006950response to stress; GO: 0007420 brain development; GO: 0007611 learningor memory

REFERENCES

-   Kelly D, Campbell J I, King T P, Grant G, Jansson E A, Coutts A G,    Pettersson S, Conway S. Commensal anaerobic gut bacteria attenuate    inflammation by regulating nuclear-cytoplasmic shuttling of    PPAR-gamma and RelA. Nat Immunol. 2004 January; 5(1):104-12.-   Xu J, Bjursell M K, Himrod J, Deng S, Carmichael L K, Chiang H C,    Hooper L V, Gordon J I. A genomic view of the human-Bacteroides    thetaiotaomicron symbiosis. Science. 2003 Mar. 28; 299(5615):2074-6.-   Wendler W M, Kremmer E, Förster R, Winnacker E L. Identification of    pirin, a novel highly conserved nuclear protein. J Biol Chem. 1997    Mar. 28; 272(13):8482-9.-   Berg, D. J., Davidson, N., Kühn, R., Müller, W., Menon, S., Holland,    G., Thompson-Snipes, L., Leach, M. W., Rennick, D. Enterocolitis and    colon cancer in interleukin-10-deficient mice are associated with    aberrant cytokine production and CD4+Th1-like responses (1996)    Journal of Clinical Investigation, 98 (4), 1010-1020.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inbiochemistry and molecular biology or related fields are intended to bewithin the scope of the following claims.

1.-71. (canceled)
 72. A method of treating or preventing an inflammatorydisorder or an autoimmune disorder comprising administering to a subjectin need thereof a therapeutically effective amount of an isolatedBacteroides species polypeptide; wherein said administering issufficient to treat or prevent the inflammatory disorder or theautoimmune disorder in the subject.
 73. The method of claim 72, whereinsaid polypeptide has at least 75% identity to a polypeptide sequence ofSEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO
 6. 74. The method of claim 72,wherein said polypeptide is Polypeptide HP.
 75. The method of claim 72,wherein said polypeptide is encoded by a polynucleotide sequence with atleast 75% identity to a polynucleotide sequence of SEQ ID NO 1, SEQ IDNO 3, or SEQ ID NO
 5. 76. The method of claim 72, wherein said isolatedBacteroides species polypeptide is expressed and purified from a hostcell.
 77. The method of claim 72, wherein said Bacteroides species isBacteroides thetaiotaomicron.
 78. The method of claim 72, wherein saidinflammatory disorder or said autoimmune disorder affects a section ofan alimentary canal, a liver, a liver cell, an epithelial cell, anepidermal cell, a neuronal cell, a kidney, a spleen, a lung, a heart, apancreas, a pancreatic cell, or any combination thereof.
 79. The methodof claim 72, wherein said administering of said therapeutically amountof said polypeptide reduces inflammation of a section of an alimentarycanal, a liver, a liver cell, an epithelial cell, an epidermal cell, aneuronal cell, a kidney, a spleen, a lung, a heart, a pancreas, apancreatic cell, or any combination thereof.
 80. The method of claim 72,wherein said inflammatory disorder or said autoimmune disorder isselected from the group consisting of an inflammatory bowel disorder(IBD), colitis, rheumatoid arthritis, psoriasis, multiple sclerosis,type I diabetes, coeliac disease, atopic dermatitis, rhinitis, irritablebowel syndrome (IBS), ulcerative colitis, pouchitis, Crohn's disease,functional dyspepsia, atopic diseases, necrotising enterocolitis,non-alcoholic fatty liver disease, gastrointestinal infection Lupus,nephritis/glomerulonephritis, asthma, COPD, myocarditis and combinationsthereof.
 81. The method of claim 72, wherein said administering of saidtherapeutically amount of said polypeptide improves intestine barrierintegrity in the subject.
 82. The method of claim 72, wherein saidadministering of said therapeutically amount of said polypeptide reducesa level of at least one type of lactose fermenting bacteria or at leastone type of non-lactose fermenting bacteria in a tissue of a subject;wherein said tissue is selected from the group consisting of amesenteric lymph node, a liver, a pancreas, a spleen, a kidney, a heart,a lung and any combination thereof.
 83. The method of claim 72, furthercomprising at least one of: preventing a reduction in a length of alarge intestine, or preventing an increase in a length of a smallintestine in the subject; and wherein said therapeutically amount ofsaid polypeptide is sufficient to maintain the length of at least oneof: the large intestine or the small intestine of the subject.
 84. Themethod of claim 72, wherein said administering of said therapeuticallyamount of said polypeptide is sufficient to: a. reduce or preventdisruption to an integrity of a mucosal epithelium; b. reduce or preventa reduction in a number of goblet cells in the mucosal epithelium; c.reduce or prevent infiltration of immune cells into a lamina propria; ord. any combination thereof.
 85. The method of claim 72, furthercomprising regulating the expression of at least one pro-inflammatorygene and/or at least one barrier integrity gene in a cell of thesubject.
 86. The method of claim 85, wherein the at least onepro-inflammatory gene or the at least one barrier integrity gene isselected from the group consisting of regenerating islet-derived 3 betagene (Reg3b), resistin-like gamma resistin like beta gene(Retnlg|Retnlb) sucrase-isomaltase (alpha-glucosidase) gene (Si),defensin alpha 24 gene (Defa24), hydroxysteroid 11-beta dehydrogenase 2gene (Hsd11b2), hydroxysteroid (17-beta) dehydrogenase 2 gene (Hsd17b2),resistin-Like Molecule-beta (RELMb), and nuclear receptor 1D1 thyroidhormone receptor alpha gene (Nr1d1|Thra).
 87. The method of claim 72,further comprising reducing an activation of a pro-inflammatory pathwayin a cell of the subject; wherein said pro-inflammatory pathway is anNF-κβ pathway.
 88. The method of claim 72, wherein said polypeptide ispresent in a pharmaceutical composition further comprising apharmaceutically acceptable excipient, diluent, or carrier.
 89. Themethod of claim 72, wherein said administering is an oral, rectal,vaginal, parenteral, intramuscular, intraperitoneal, intra-arterial,intrathecal, intrabronchial, subcutaneous, intradermal, intravenous,nasal, buccal or sublingual administering.
 90. A pharmaceuticalcomposition that comprises: a therapeutically effective amount of anisolated polypeptide, wherein said isolated polypeptide is apirin-related protein or a derivative thereof; and a pharmaceuticallyacceptable excipient, diluent or carrier.
 91. The pharmaceuticalcomposition of claim 90, wherein said isolated polypeptide has at least75% identity to a polypeptide sequence of SEQ ID NO 2, SEQ ID NO 4 orSEQ ID NO
 6. 92. The pharmaceutical composition of claim 90, whereinsaid isolated polypeptide is Polypeptide HP.
 93. The pharmaceuticalcomposition of claim 90, wherein said isolated polypeptide isencapsulated.
 94. The pharmaceutical composition of claim 93, whereinsaid pharmaceutical composition further comprises an enteric coating.95. The pharmaceutical composition of claim 90, wherein said isolatedpolypeptide is a recombinant polypeptide.
 96. The pharmaceuticalcomposition of claim 90, wherein said isolated polypeptide is expressedand purified from a host cell.
 97. The pharmaceutical composition ofclaim 90, wherein said pharmaceutical composition is in unit dose form.98. A method of making a pharmaceutical composition comprising admixinga therapeutically effective amount of a polypeptide with at least 75%identity to a polypeptide sequence of SEQ ID NO 2, SEQ ID NO 4 or SEQ IDNO 6 with a pharmaceutically acceptable excipient, carrier or diluent.99. The method of claim 98, wherein said polypeptide is encapsulated.100. The method of claim 98, wherein said polypeptide is recombinantproduced prior to admixing with said pharmaceutically acceptableexcipient, carrier, or diluent.
 101. The method of claim 98, whereinsaid polypeptide is encoded by a polynucleotide sequence with at least75% identity to a polynucleotide sequence of SEQ ID NO 1, SEQ ID NO 3,or SEQ ID NO 5.